JP6805428B2 - Humanized antibody against CEACAM1 - Google Patents

Description

The present invention primarily relates to humanized antibodies capable of specifically binding to human CEACAM molecules. More specifically, the present invention relates to antibodies against CEACAM1 that include mouse-derived CDRs as well as humanized heavy and light chain regions with specific reversion mutations.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), also known as cluster of differentiation 66a (CD66a), is a member of the carcinoembryonic antigen (CEA) gene family and immunoglobulin (Ig). ) Belongs to the super family. CEACAM1 is upregulated in T cells and NK cells upon activation, and its allophilic interaction leads to inhibition of the cytotoxic effects of lymphocytes. Studies of several human tumor types suggest that the use of the CEACAM1 pathway can enable tumor-induced immune evasion. Preclinical animal models of tumors have shown that blocking CEACAM1 interactions with monoclonal antibodies (mAbs) can enhance the immune response to tumors. In 2013, there were an estimated 1,660,290 new cancer cases and 580,350 cancer-related deaths in the United States.

Blocking checkpoint immunotherapy has been shown to be an exciting new idea in cancer treatment. The immune checkpoint pathway consists of a range of co-stimulatory and inhibitory molecules that work together to maintain self-tolerance and protect tissues from damage by the immune system under physiological conditions. Tumors utilize specific checkpoint pathways to evade the immune system. Therefore, inhibition of such pathways has emerged as a promising anti-cancer therapeutic strategy (Pardol, DM, 2012, Nat Rev Cancer, 12, 252-264). Anti-tumor immunotherapy via CEACAM1 blockade is not limited to any single tumor type in principle, but has the activity of enhancing the therapeutic immune response against several histologically different tumors. Can have.

The anti-cytotoxic T lymphocyte 4 (CTLA-4) antibody ipilimumab (approved in 2011) was the first immunotherapeutic agent to show benefits for the treatment of cancer patients (Robert et al., 2011). , N. Engl. J. Med., Vol. 364, pp. 2517-2526). This antibody interferes with the inhibitory signal during antigen presentation to T cells. Anti-programmed cell death 1 (PD-1) antibody pembrolizumab (approved in 2014) blocks the negative immunomodulatory signals of PD-1 receptors expressed by T cells (Hamid, 2013, N. Engl. J. Med., Vol. 2, pp. 134-144). Blocking PD-1 / PL-L1 axis antibodies has shown promising results in several clinical trials in patients with various tumor types (Dolan and Gupta 2014, Cancer Control, Vol. 21, Pages 231-237). An additional anti-PD-1 agent was submitted for regulatory approval in 2014 for the treatment of non-small cell lung cancer (NSCLC). Currently, there are active studies investigating many other immune checkpoints, including lymphocyte activation gene 3 (LAG3), CD 137, OX40 (also called CD134), and killer cell immunity. There are globulin-like receptors (KIRs) (Gelao et al., 2014, Toxins, Vol. 6, pages 914-933).

A humanized antibody is a non-human species antibody (eg, a mouse antibody) whose protein sequence has been modified to enhance similarity to naturally produced antibody variants in humans. The process of "humanization" is usually applied to monoclonal antibodies developed for administration to humans, where the process of developing specific antibodies involves production in a non-human (such as mouse) immune system. Is executed. The protein sequence of the antibody thus produced is different from naturally occurring antibodies in humans and is therefore immunogenic when administered to human patients. Humanized antibodies have a protein sequence similar to human antibodies, but are believed to differ from chimeric antibodies, which carry large stretches of non-human proteins.

It is possible to produce humanized antibodies without producing chimeric intermediates. Direct production of humanized antibodies can be achieved by inserting appropriate CDR coding segments (responsible for the desired binding properties) into the scaffolding of human antibodies, a process known as "CDR transplantation". .. In general, after an antibody has been developed to have the desired properties in a mouse (or another non-human animal), the sequence of DNA encoding the CDR of that antibody can be determined. Once the exact sequences of the desired CDRs are known, these sequences are inserted into the DNA-containing constructs of the human antibody framework.

WO 2010/125571 of the present inventors discloses a mouse monoclonal antibody (MRG-1) produced by a specific hybridoma cell. mAbs are highly selective for CEACAM1 and do not cross-react with other members of the CEACAM family. WO 2013/054331 of the present inventors also discloses a highly selective chimeric antibody (CM-10) for CEACAM1.

Although progress has been made in the field of immunotherapy, it is more effective, longer lasting, with new targets, as a signal agent, or known to ultimately result in a long-lasting long-term response in cancer patients. There is a constant need for new therapeutic agents that can function in combination with the above therapies. The need to provide a humanized antibody that recognizes a particular CEACAM protein that is safer, more potent, and can be used in the diagnosis and treatment of diseases involving the expression or activation of the CEACAM protein has not been met.

The present invention provides a humanized antibody that recognizes CEACAM1. The selected humanized antibody of the present invention contains a number of specific "return mutations" in its variable region sequence, namely mutations reverted from the humanized sequence to the mouse sequence. These reversions occur in residues important for maintaining the conformation and binding affinity of the original antibody and have the lowest incidence of potential T cell epitopes, resulting in a detrimental immune response to the antibody. Minimize risk.

In order to produce humanized mAbs that recognize CEACAM1 with a particular CDR sequence in the desired orientation and configuration and human framework, the inventors of the present invention have significant residues that influence CDR presentation. Is identified within the human framework (outside the CDR sequence) and a series of mutations within these important residues to restore the correct presentation of the CDR while minimizing the immunogenicity of the antibody. Designed. Therefore, for the first time, the present invention provides high affinity, non-immunogenic, highly specific humanized antibodies to CEACAM1.

According to one aspect, the invention specifically recognizes human CEACAM1 and comprises a humanized monoclonal antibody (mAb) comprising at least an antigen binding domain and at least one variable region selected from the group consisting of: Fragments are provided: (i) heavy chain variable regions containing CDR1, CDR2 and CDR3 containing the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and (ii) SEQ ID NO: 4, SEQ ID NO: 3, respectively. A light chain variable region comprising CDR1, CDR2 and CDR3 comprising the amino acid sequence set forth in 5 and SEQ ID NO: 6; at least one of (i) and (ii) thereof is 1 to 25 from the human sequence to the mouse sequence. Contains reversion mutations of a single amino acid residue.

According to some embodiments, the present invention provides a humanized monoclonal antibody (mAb) or fragment thereof that specifically recognizes human CEACAM1 and comprises at least one variable region selected from the group consisting of: : (I) A heavy chain variable region amino acid sequence containing the CDR sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the heavy chain variable region amino acid sequence therein is 1 to 25 in the framework sequence. Amino acid residues differ from SEQ ID NO: 57; and (ii) a light chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and a light chain variable region amino acid sequence therein. The 1 to 10 amino acid residues in the framework sequence differ from SEQ ID NO: 58.

In certain embodiments, the present invention specifically recognizes human CEACAM1 and provides a humanized monoclonal antibody (mAb) or fragment thereof comprising: (i) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 , CDR sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; (ii) heavy chain variable region amino acid sequences in which 3 to 13 framework amino acid residues differ from SEQ ID NO: 57; and (iii) 3 to. A light chain variable region amino acid sequence in which the five framework amino acid residues differ from SEQ ID NO: 58.

According to a particular embodiment, the heavy chain variable region amino acid sequence differs from SEQ ID NO: 57 in 3 to 13 amino acid residues in the framework sequence. According to another embodiment, the light chain variable region amino acid sequence differs from SEQ ID NO: 58 in 3-5 amino acid residues in the framework sequence.

According to other embodiments, the present invention (i) comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the amino acids set forth in SEQ ID NO: 9. Heavy chain variable regions containing framework amino acid sequences in which the sequences differ from 2-9 amino acids; and / or (ii) CDR1, CDR2 and / or (ii) containing the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. A non-completely humanized monoclonal antibody containing CDR3 and containing a light chain variable region containing a framework amino acid sequence in which 2 to 4 amino acids differ from the amino acid sequence set forth in SEQ ID NO: 13 and human CEACAM1 are specifically recognized. The analogs, derivatives, and antigen-binding fragments thereof are provided.

According to some embodiments, the mAb sequence comprises 1 to 50 reversion mutations to the mouse sequence. According to other embodiments, the mAb sequence comprises 2-30 reversion mutations to the mouse sequence. According to yet another embodiment, the mAb sequence comprises 3-20 reversion mutations to the mouse sequence. According to other embodiments, the mAb sequence comprises 4-15 reversion mutations to the mouse sequence. According to other embodiments, the heavy chain of the mAb sequence contains 1 to 15 reversion mutations to the mouse sequence. According to other embodiments, the light chain of the mAb sequence comprises 1 to 15 reversion mutations to the mouse sequence. According to other embodiments, the heavy chain of the mAb sequence contains 2-9 reversion mutations to the mouse sequence and the light chain of the mAb sequence contains 2-4 reversion mutations to the mouse sequence. Including.

According to some specific embodiments, the humanized mAb comprises 1-25 mutations from the human sequence to the mouse sequence in the heavy chain sequence set forth in SEQ ID NO: 57. According to some embodiments, the heavy chain of the humanized mAb is a group consisting of V11, R38, M48, V68, M70, R72, T74, S77, R85, R87, T91, Y95 and T115 of SEQ ID NO: 57. The residue selected from contains at least one mutation. According to some embodiments, at least one return mutation in SEQ ID NO: 57 consists of the group consisting of V11L, R38K, M48I, V68A, M70L, R72A, T74K, S77N, R85S, R87T, T91S, Y95F and T115S. Be selected.

According to some other specific embodiments, the humanized mAb comprises 1-25 mutations from the human sequence to the mouse sequence in the light chain sequence set forth in SEQ ID NO: 58. According to some embodiments, the light chain of the humanized mAb comprises at least one mutation in a residue selected from the group consisting of P44, F71, F73, P80 and Y87 of SEQ ID NO: 58. According to some embodiments, at least one reversion mutation in SEQ ID NO: 58 is selected from the group consisting of P44V, F71Y, F73L, P80Q, and Y87F.

According to some embodiments, the heavy chain of the humanized mAb is a group consisting of V11, R38, M48, V68, M70, R72, T74, S77, R85, R87, T91, Y95 and T115 of SEQ ID NO: 57. The light chain of the humanized mAb comprises at least one mutation in the residue selected from, and the light chain of the humanized mAb is at least one in the residue selected from the group consisting of P44, F71, F73, P80, and Y87 of SEQ ID NO: 58. Includes mutations. Each possibility represents another embodiment of the invention.

According to some embodiments, the heavy chain of the humanized mAb is a residue selected from the group consisting of V68, M70, R72, T74, S77, R85, R87, T91, and Y95 of SEQ ID NO: 57. Containing at least one mutation, the light chain of the humanized mAb comprises at least one mutation in a residue selected from the group consisting of F71, F73, P80 and Y87 of SEQ ID NO: 58. Each possibility represents another embodiment of the invention.

According to some embodiments, at least one reversion mutation of SEQ ID NO: 57 is selected from the group consisting of V11L, R38K, M48I, V68A, M70L, R72A, T74K, S77N, R85S, R87T, T91S, Y95F and T115S. And at least one reversion of SEQ ID NO: 58 is selected from the group consisting of P44V, F71Y, F73L, P80Q, and Y87F. Each possibility represents another embodiment of the invention.

According to some embodiments, at least one reversion mutation of SEQ ID NO: 57 is selected from the group consisting of V68A, M70L, R72A, T74K, S77N, R85S, R87T, T91S, and Y95F, and at least one of SEQ ID NO: 58. One return mutation is selected from the group consisting of F71Y, F73L, P80Q, and Y87F. Each possibility represents another embodiment of the invention.

In some embodiments, the monoclonal antibody comprises SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 12, and SEQ ID NO: 23. Includes at least one heavy chain framework sequence described in the sequence selected from the group. In some embodiments, the monoclonal antibody comprises the heavy chain framework sequence set forth in a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22. Each possibility represents another embodiment of the invention.

In some embodiments, the monoclonal antibody is SEQ ID NO: 7 or SEQ ID NO: 15; SEQ ID NO: 16 or SEQ ID NO: 17; SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22; and SEQ ID NO: Includes the heavy chain framework sequence set forth in number 10 or SEQ ID NO: 23. Each possibility represents another embodiment of the invention.

In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in a sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32. Each possibility represents another embodiment of the invention. In some specific embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 32.

In some embodiments, the monoclonal antibody comprises the light chain framework sequence set forth in a sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

In some embodiments, the monoclonal antibody comprises the light chain framework sequence set forth in a sequence selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27. Each possibility represents another embodiment of the invention.

In some embodiments, the monoclonal antibody is the light chain frame according to a sequence selected from the group consisting of SEQ ID NO: 11; SEQ ID NO: 24; SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27; and SEQ ID NO: 14. Includes work array. Each possibility represents another embodiment of the invention.

In some embodiments, the monoclonal antibody comprises the light chain variable region sequence set forth in a sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35. Each possibility represents another embodiment of the invention. In some specific embodiments, the monoclonal antibody comprises the light chain variable region sequence set forth in SEQ ID NO: 34.

In certain embodiments, the monoclonal antibody is (i) SEQ ID NO: 7 or SEQ ID NO: 15; SEQ ID NO: 16 or SEQ ID NO: 17; SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22; And the heavy chain framework sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 23, and (ii) SEQ ID NO: 11; SEQ ID NO: 24; SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27; and the light chain set forth in SEQ ID NO: 14. Contains a framework array. Each possibility represents another embodiment of the invention.

In some embodiments, the monoclonal antibody is the heavy chain variable region sequence set forth in SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32; and SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: Includes the light chain variable region sequence of number 35. Each possibility represents another embodiment of the invention. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 32 and the light chain variable region sequence set forth in SEQ ID NO: 34.

In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 28 and the light chain variable region sequence set forth in SEQ ID NO: 33. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 29 and the light chain variable region sequence set forth in SEQ ID NO: 33. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 30 and the light chain variable region sequence set forth in SEQ ID NO: 33. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 31 and the light chain variable region sequence set forth in SEQ ID NO: 33. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 32 and the light chain variable region sequence set forth in SEQ ID NO: 33.

In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 28 and the light chain variable region sequence set forth in SEQ ID NO: 34. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 29 and the light chain variable region sequence set forth in SEQ ID NO: 34. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 30 and the light chain variable region sequence set forth in SEQ ID NO: 34. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 31 and the light chain variable region sequence set forth in SEQ ID NO: 34.

In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 28 and the light chain variable region sequence set forth in SEQ ID NO: 35. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 29 and the light chain variable region sequence set forth in SEQ ID NO: 35. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 30 and the light chain variable region sequence set forth in SEQ ID NO: 35. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 31 and the light chain variable region sequence set forth in SEQ ID NO: 35. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 32 and the light chain variable region sequence set forth in SEQ ID NO: 35.

According to some embodiments, the heavy chain of the humanized mAb is selected from IgG4 and IgG1 isotypes. According to some embodiments, the heavy chain of the humanized mAb is an IgG4 isotype. According to some embodiments, the light chain of the human mAb is a kappa isotype. According to some specific embodiments, the humanized mAb comprises a light chain kappa isotype and a heavy chain IgG4 isotype. According to other specific embodiments, the humanized mAb comprises a light chain kappa isotype and a heavy chain IgG1 isotype.

According to some embodiments, the mAb comprises the light chain set forth in SEQ ID NO: 52. According to some embodiments, the mAb comprises the heavy chain set forth in SEQ ID NO: 53 or SEQ ID NO: 59. According to some embodiments, the mAb comprises the light chain set forth in SEQ ID NO: 52 and the heavy chain set forth in SEQ ID NO: 53. According to some embodiments, the mAb comprises the light chain set forth in SEQ ID NO: 52 and the heavy chain set forth in SEQ ID NO: 59.

According to some embodiments, the mAb comprises the light chain variable region set forth in SEQ ID NO: 58 and the heavy chain set forth in SEQ ID NO: 53. According to some embodiments, the mAb comprises the light chain variable region set forth in SEQ ID NO: 58 and the heavy chain set forth in SEQ ID NO: 59. According to some embodiments, the mAb comprises a light chain set forth in SEQ ID NO: 52 and a heavy chain variable region set forth in SEQ ID NO: 57.

The present invention also provides a mAb comprising the light chain variable region set forth in SEQ ID NO: 58 and the heavy chain variable region set forth in SEQ ID NO: 57.

In some embodiments, the humanized mAb or antigen-binding fragment thereof can bind to the human CEACAM1 protein with an affinity of at least about ^10-8 M. In some embodiments, the humanized mAb or antigen-binding fragment thereof is capable of binding to at least one of human CEACAM 3 and human CEACAM 5 proteins with an affinity of at least about 5 × ^10-7 M.

In yet another aspect, the invention provides analogs and / or derivatives of the monoclonal antibody that have at least 90% sequence identity with the antigen-binding fragment of the monoclonal antibody.

Fragments of mAbs that recognize CEACAM1 and contain at least one antigen binding domain are also included within the scope of the invention as long as they contain at least one reversion mutation from the CDR sequence defined above and the human sequence to the mouse sequence. ..

In yet another aspect, the invention provides an isolated polynucleotide or fragment thereof encoding the above monoclonal antibody that specifically recognizes human CEACAM1.

In some embodiments, the isolated polynucleotide sequence is the DNA sequence set forth in any one of SEQ ID NOs: 44-51 encoding the humanized mAb variable region, or at least 90% sequence identity with the above sequence. Includes its analogs with. In some embodiments, the isolated polynucleotide sequence has at least 90% sequence identity with the DNA sequence set forth in SEQ ID NO: 54 or SEQ ID NO: 55 encoding the heavy strand of humanized mAb, or the above sequence. Includes its analogs. In some embodiments, the isolated polynucleotide sequence comprises the DNA sequence set forth in SEQ ID NO: 56 encoding the light chain of humanized mAb, or an analog thereof having at least 90% sequence identity with the above sequence. Including.

The present invention further provides, in another aspect, a plasmid containing the isolated polynucleotide described above.

In yet another aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the above monoclonal antibody and a pharmaceutically acceptable carrier, diluent or excipient.

According to some embodiments, the pharmaceutical composition comprises humanized mAbs to 1-50 mg / ml CEACAM1. According to some embodiments, the pharmaceutical composition comprises a basic amino acid. According to some embodiments, the pharmaceutical composition comprises sugar. According to some embodiments, the pharmaceutical composition comprises a surfactant. According to some embodiments, the pharmaceutical composition comprises basic amino acids, sugars and surfactants. According to some embodiments, the pharmaceutical composition comprises (i) 1-10 mg / ml basic amino acids, (ii) 10/100 mg / ml sugars, (iii) 0.01-1 mg / ml. Surfactant, humanized mAb to (iv) 1-50 mg / ml CEACAM1, 4-6 mg / ml basic amino acids, 70-100 mg / ml sugar and 0.1-1 mg / ml non-anionic surface activity The agent, or (v) humanized mAb to 10 mg / ml CEACAM1, 4.65 mg / ml L-histidine, 82 mg / ml sucrose, and 0.20 mg / ml polysorbate 20.

According to some embodiments, the basic amino acid is selected from the group consisting of histidine, arginine, lysine and ornithine. Each possibility represents another embodiment of the invention. According to some embodiments, the composition comprises 1-10, 2-9, 3-7 or 4-6 mg / ml of basic amino acids. Each possibility represents another embodiment of the invention.

According to some embodiments, the sugar is selected from the group consisting of sucrose, trehalose, glucose, dextrose and maltose. Each possibility represents another embodiment of the invention. According to some embodiments, the composition comprises 10-200, 10-100, 50-150 or 70-100 mg / ml sugar. Each possibility represents another embodiment of the invention.

According to yet another embodiment, the composition comprises a polyol comprising, but not limited to, mannitol and sorbitol. Each possibility represents another embodiment of the invention.

According to some embodiments, the surfactant is non-anionic. According to some embodiments, the surfactant is selected from the group consisting of polysorbate, sorbitan ester and poloxamer. Each possibility represents another embodiment of the invention. According to some embodiments, the surfactant is selected from the group consisting of polysorbate 20 and polysorbate 80. Each possibility represents another embodiment of the invention. According to some embodiments, the composition comprises 0.01-10, 0.01-1, 0.05-5 or 0.1-1 mg / ml surfactant. Each possibility represents another embodiment of the invention. According to some embodiments, the pharmaceutical composition comprises 4-6 mg / ml basic amino acids, 70-100 mg / ml sugars and 0.1-1 mg / ml surfactant.

According to some embodiments, the pharmaceutical composition is in liquid form and comprises 1-50 mg / ml humanized mAbs to CEACAM1 containing at least one reversion to the mouse sequence. According to other embodiments, the pharmaceutical composition is lyophilized. According to some embodiments, the pharmaceutical composition comprises humanized mAb to 10 mg / ml CEACAM1 and 4.65 mg / ml L-histidine, 82 mg / ml sucrose and 0.20 mg / ml polysorbate 20. Including.

According to some embodiments, the pharmaceutical composition comprises at least one humanized mAb or fragment as defined above, and an additional immunomodulatory or kinase inhibitor. According to some embodiments, the pharmaceutical composition comprising at least one humanized mAb or fragment as defined above, and the pharmaceutical composition comprising an additional immunomodulatory or kinase inhibitor, may be administered separately. Used to treat cancer.

According to some specific embodiments, the additional immunomodulatory substance is an anti-human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2 antibody, activated cytotoxic lymphocytes. It is selected from the group consisting of cells, lymphocyte activators, and RAF / MEK pathway inhibitors. Each possibility represents another embodiment of the invention. According to some specific embodiments, the additional immunomodulatory substance is mAb for PD-1, mAb for PD-L1, mAb for PD-L2, interleukin 2 (IL-2), lymphokine activation. Selected from the group consisting of killer (LAK) cells.

According to some embodiments, the present invention comprises a pharmaceutical composition comprising a humanized mAb or an antigen-binding fragment thereof containing a reversion mutation as defined above, for use in the treatment of cancer by separate administration. Also provided are pharmaceutical compositions comprising a mAb or an antigen-binding fragment thereof for at least one of human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2.

According to another embodiment, a pharmaceutical composition comprising a humanized mAb or an antigen-binding fragment thereof for CEACAM as defined above and activated cytotoxic lymphocytes is provided. In certain embodiments, the activated cytotoxic lymphocytes are selected from the group consisting of LAK cells, CIK cells, and any combination thereof. Each possibility represents another embodiment of the invention. In certain embodiments, the activated cytotoxic lymphocytes are lymphokine-activated killer (LAK) cells.

In other embodiments, the pharmaceutical composition comprises a humanized mAb to CEACAM1 or an antigen-binding fragment thereof, and a lymphocyte activator or fragment thereof, an analog or a fusion protein. In certain embodiments, the lymphocyte activator is selected from the group consisting of IL-2, IFNγ, anti-CD3 antibodies and fragments thereof, analogs or fusion proteins. In certain embodiments, the lymphocyte activator is IL-2 or a fragment, analog or fusion protein thereof. Each possibility represents another embodiment of the invention.

In yet another embodiment, the pharmaceutical composition is selected from the group consisting of B-Raf kinase variants, MEK1 and MEK2 kinases, and humanized mAbs to human CEACAM1 as defined above or antigen-binding fragments thereof. Contains kinase inhibitors.

According to some specific embodiments, the B-Raf kinase inhibitor attenuates or blocks the phosphorylation of MEK1 or MEK2 by the B-Raf kinase variant. In certain embodiments, the B-Raf kinase inhibitor attenuates or blocks the dimerization of the B-Raf kinase variant. In certain embodiments, MEK1 kinase inhibitors attenuate or block the phosphorylation of MAPK by MEK1 kinase. In certain embodiments, MEK2 kinase inhibitors attenuate or block the phosphorylation of MAPK by MEK2 kinase.

The present invention further according to other embodiments are B-Raf kinase variants for use in the treatment of cancer, kinase inhibitors selected from the group consisting of MEK1 kinase and MEK2 kinase, and humans against human CEACAM1. Provided is a conjugated mAb or an antigen-binding fragment thereof. The two active ingredients can be part of one pharmaceutical composition or separate pharmaceutical compositions that can be administered simultaneously or separately.

The humanized mAb and additional immunomodulatory material for CEACAM1 of the present invention can be contained in one pharmaceutical composition or in separate compositions for simultaneous or separate administration.

In some embodiments, the pharmaceutical composition is for the treatment of a disease or disorder associated with the expression, activation or function of a CEACAM protein family member, including but not limited to CEACAM1. Each possibility represents another embodiment of the invention.

In some embodiments, the pharmaceutical composition is for the treatment of a disease or disorder associated with expression, activation or function of CEACAM1. In some embodiments, the disease or disorder is a cell proliferative disease or disorder. Each possibility represents another embodiment of the invention. In some embodiments, the cell proliferative disorder or disorder is cancer.

According to some embodiments, the cancers are melanoma, colorectal cancer, bladder cancer, lung cancer, non-small cell lung cancer (NSCLC), non-small cell lung adenocarcinoma (NSCLA), gastrointestinal cancer, pancreatic cancer, breast cancer. , Prostate cancer, thyroid cancer, gastric cancer, ovarian cancer, myeloma and uterine cancer. Each possibility represents another embodiment of the invention.

In yet another aspect, the present invention provides a diagnostic composition comprising at least one humanized mAb or fragment thereof that specifically recognizes human CEACAM1 as described above.

In yet another aspect, the invention is a method of preventing, attenuating, or treating a disease or disorder associated with the expression, activation, or function of the CEACAM1 protein, wherein the therapeutically effective amount of the pharmaceutical composition described above. Provided are methods that include administering a substance to a subject in need thereof.

In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is an infectious disease, such as a viral infection.

In some embodiments, the isolated antibody contained in the pharmaceutical composition is bound to a cytotoxic moiety.

In certain embodiments, the method comprises administering to the subject at least one dose of humanized mAb relative to CEACAM1 in the range of 0.01 mg / kg to 10 mg / kg body weight. In certain embodiments, the above methods include (i) the same or different multiple doses of humanized mAbs, (ii) multiple incremental doses; or (iii) the pharmaceutical composition once a week for two weeks. It includes administration once, once every three weeks, once every four weeks, or once every five weeks. In certain embodiments, the method described above comprises 1 to 10 dosing cycles, each cycle comprising 2 to 5 infusions of humanized mAb every 1 to 4 weeks, and 2 during each cycle. There is an interval of ~ 8 weeks.

In certain embodiments, the above method further comprises administration of one lymphocyte cell or multiple lymphocyte cells. In some embodiments, the method further comprises administering CEACAM1-expressing lymphocytes to the subject. In some embodiments, the lymphocytes include T cells, NK cells or tumor infiltrating lymphocytes (TIL). Each possibility represents another embodiment of the invention. In some embodiments, the lymphocyte cells express CEACAM1, PD-1, or both. Each possibility represents another embodiment of the invention. In certain such embodiments, the lymphocyte cells express CEACAM1 and PD-1. In some embodiments, the lymphocyte cells are tumor infiltrating lymphocytes (TIL) cells, lymphokine-activated killer (LAK) cells, cytokine-induced killer (CIK) cells, T cells, B cells, NK cells, and those. Selected from the group consisting of any combination of. Each possibility represents another embodiment of the invention. In certain such embodiments, the lymphocyte cells are selected from the group consisting of tumor infiltrating lymphocytes (TIL) cells and lymphokine-activated killer (LAK) cells. In certain such embodiments, the lymphocyte cells are one tumor infiltrating lymphocyte (TIL) cell or multiple TIL cells. In certain such embodiments, the lymphocyte cells are a single lymphokine-activated killer (LAK) cell or a plurality of LAK cells. In certain embodiments, lymphocyte cells are activated. In some embodiments, lymphocyte cells are cytotoxic to cancer cells. In some embodiments, the cancer cells express CEACAM1, PD-L1, PD-L2, or any combination thereof. Each possibility represents another embodiment of the invention. In some embodiments, the cancer cells express CEACAM1, PD-L1, or both. Each possibility represents another embodiment of the invention. In some embodiments, the cancer cells express CEACAM1, PD-L2, or both. Each possibility represents another embodiment of the invention. In certain such embodiments, the cancer cells express CEACAM1, PD-L1 and PD-L2.

In certain embodiments, the method further comprises administering to the subject a lymphocyte activator. According to some embodiments, the lymphocyte activator is selected from the group consisting of IL-2, IFNγ, and anti-CD3 antibodies. In certain embodiments, the above method further comprises administering to the subject an additional anti-cancer composition.

The present invention further provides, in another aspect, a method of immunomodulation comprising contacting the above antibody or fragment thereof with CEACAM1-expressing lymphocytes.

In yet another embodiment, the present invention is a method of inhibiting the migration of CEACAM1 expressing tumor cells, wherein the antibody or fragment thereof is brought into contact with the CEACAM1 expressing tumor cells, thereby causing the migration of the CEACAM1 expressing tumor cells. Provide methods that include inhibiting.

In yet another embodiment, the present invention is a method of inhibiting the homozygous or atypical protein-protein interaction of CEACAM1 in which the above antibody or fragment thereof is brought into contact with CEACAM1 expressing lymphocytes, thereby isomorphic or atypical of CEACAM1. Provided are methods that include inhibiting protein-protein interactions.

In yet another embodiment, the invention is a method of increasing the duration or progression of a subject having cancer, the subject being an effective amount of a composition comprising the above monoclonal antibody, and an antitumor composition. The anti-tumor composition comprises at least one chemotherapeutic agent, whereby co-administration of the antibody and the anti-tumor composition effectively increases the duration or progression of survival. Provide a method including.

The methods of the invention may include administering the pharmaceutical composition as defined above with an additional anti-cancer composition. According to certain embodiments, the anti-cancer composition comprises at least one chemotherapeutic agent. According to other specific embodiments, the anti-cancer composition comprises an immunomodulator. Anti-cancer agents that can be administered with or separately from the antibodies of the invention may include any such agent known in the art that exhibits anti-cancer activity.

According to some embodiments, a method of treating cancer, a pharmaceutical composition comprising a humanized antibody that recognizes CEACAM1 and contains a reversion to a mouse sequence, and an additional immunomodulatory or kinase inhibitor. Provided are methods comprising administering to a subject in need thereof a pharmaceutical composition comprising.

According to some embodiments, the immunomodulatory substance is a mAb for PD-1, mAb for PD-L1, mAb for PD-L2, interleukin 2 (IL-2), lymphokin activation killer (LAK) cells. Selected from the group consisting of, the kinase inhibitor is a B-Raf / MEK inhibitor.

In some embodiments, the method comprises administration of two or more pharmaceutical compositions. In some embodiments, administration of two or more of the pharmaceutical compositions is carried out simultaneously. In some embodiments of the method, administration of two or more of the pharmaceutical compositions is performed sequentially. In some embodiments of the method, additional immunomodulatory material is administered prior to humanized mAbs to human CEACAM1 or antigen-binding fragments thereof. In some embodiments of the method, the immunomodulatory substance is administered simultaneously with mAb or an antigen-binding fragment thereof to human CEACAM1. In some embodiments of the method, the immunomodulatory substance is administered after humanized mAbs to human CEACAM1 or antigen-binding fragments thereof.

In some embodiments, the method administers to the patient a pharmaceutical composition comprising a monoclonal antibody against human CEACAM1 or an antigen-binding fragment thereof, and a pharmaceutical composition comprising a monoclonal antibody against human PD-1 or an antigen-binding fragment thereof. Including to do. Each possibility represents another embodiment of the invention.

According to still another embodiment, the above method of treating a patient with cancer comprises a pharmaceutical composition comprising an inhibitor of a kinase selected from the group consisting of B-Raf kinase variants, MEK1 kinase and MEK2 kinase. In addition, a pharmaceutical composition containing a monoclonal antibody against human CEACAM1 or an antigen-binding fragment thereof is administered to the patient, and the cancer cells express a B-Raf kinase variant to treat the cancer.

In some embodiments, the above method for treating cancer further comprises administering to the patient a pharmaceutical composition comprising lymphocytes. In some embodiments, administration of the pharmaceutical composition comprising lymphocyte cells is carried out simultaneously with at least one of the pharmaceutical compositions comprising the antibody. In some embodiments of the method, administration of two or more pharmaceutical compositions is performed sequentially.

In some embodiments, lymphocyte cells are pre-incubated with humanized mAbs to human CEACAM1 and antigen-binding fragments thereof, or with additional immunomodulators. Each possibility represents another embodiment of the invention.

The pharmaceutical composition of the present invention is administered by any suitable means such as intranasal administration, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intra-articular administration, intralesional administration, oral administration, or local administration. Can be done. According to some embodiments, the pharmaceutical composition is administered parenterally. According to some specific embodiments, intravenous (iv) administration is utilized.

The methods of the invention for treating cancer or other CEACAM1-related diseases or disorders, according to some embodiments, include at least one dose of humanized mAb relative to CEACAM1 in the range of 0.01 mg / kg to 10 mg / kg body weight. , Including administration to subjects in need of it.

According to some embodiments, this at least one dose is selected from the group consisting of 0.01-0.1 mg / kg, 0.1-1 mg / kg; 1-10 mg / kg; and 10-50 mg / kg. Will be done.

According to some embodiments, the method comprises administration of multiple doses of humanized mAb, the multiple doses being the same or different. According to some embodiments, the method comprises administering a plurality of escalating doses. According to some embodiments, the method comprises at least one cycle of administration for at least 12 weeks.

According to other embodiments, the treatment period is 12-50 weeks. According to some specific embodiments, the treatment period is selected from the group consisting of 12-20 weeks, 20-30 weeks and 30-50 weeks. According to yet another embodiment, the treatment regimen comprises several dosing cycles, each at least 12 weeks.

According to some embodiments, the therapeutic regimen comprises 1-8 cycles, each cycle comprising 4 infusions of humanized anti-CEACAM mAb at least 4 weeks. According to some embodiments, the treatment regimen comprises 2-6 cycles.

According to some embodiments, administration is once a week, once every two weeks, once every three weeks, once every four weeks, or once every five weeks. Each possibility represents another embodiment of the invention.

According to some embodiments, the treatment regimen comprises 1-10 cycles, each cycle comprising 2-5 infusions of the humanized mAb of the invention every 1-4 weeks, during each cycle. There is an interval of 2 to 8 weeks.

According to some embodiments, administration starts at 0.01 mg / kg and continues with 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, and 10 mg / kg. A dose escalation regimen containing is provided. According to yet another embodiment, the treatment regimen comprises 6 cycles of 4 infusions, each administered every 2 weeks.

In yet another aspect, the present invention is a method of diagnosing a cancer of a subject in need thereof, in which a biological sample derived from or obtained from the subject is brought into contact with the diagnostic composition. Provided is a method in which complex formation exceeding a predetermined threshold value is an index of the cancer of the subject.

The present invention further provides, in another embodiment, a method of determining the expression of CEACAM1 comprising contacting the antibody or fragment thereof with a biological sample and measuring the level of immune complex formation. To do. According to some embodiments, the method comprises comparing the level of the immune complex with a standard curve obtained from a known amount of CEACAM1.

In yet another embodiment, the invention is a method of diagnosing a disease or disorder associated with CEACAM protein expression, the step of incubating a monoclonal antibody with a biological sample as described above; binding using a detectable probe. Step of detecting CEACAM protein; step of comparing the amount of bound CEACAM protein with a standard curve obtained from a reference sample containing a known amount of CEACAM protein; calculating the amount of CEACAM protein in a biological sample from the standard curve. Steps; and provide a method comprising the step of comparing the amount of normal CEACAM protein with the amount of CEACAM protein.

According to some embodiments, the humanized mAbs of the invention are used as predictive biomarkers associated with anti-CEACAM1 treatment based on the expression level of CEACAM1 in pretreatment tumor specimens. Expression of CEACAM1 levels is determined using methods known in the art utilizing the humanized mAbs of the invention.

The present invention further provides, in one aspect, the use of the above-mentioned monoclonal antibodies for the diagnosis, prevention or treatment of diseases or disorders or infections associated with cell proliferation or angiogenesis.

The present invention further provides, in one aspect, the use of the above-mentioned monoclonal antibodies for the manufacture of a medicament for the treatment of disorders or diseases associated with the expression or activity of the CEACAM protein.

The present invention further provides, in one aspect, the use of the above-mentioned monoclonal antibodies for the preparation of diagnostic compositions for the diagnosis of diseases or disorders or infections associated with cell proliferation or angiogenesis.

The present invention further, in one embodiment, is a heavy chain variable region comprising the framework amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 or SEQ ID NO: 25, SEQ ID NO: 26. Alternatively, a partially humanized monoclonal antibody comprising a light chain variable region comprising the framework amino acid sequence set forth in SEQ ID NO: 27 is provided.

Further embodiments of the present invention and the full scope of applicability will become apparent from the detailed description below. However, since various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from the mode for carrying out the invention, the mode and embodiment for carrying out the invention showing the preferred embodiment of the present invention. The example should be understood to be given as an example only.

It is a structural model of the chimeric anti-CEACAM-1 antibody (CM-10) V region of the top view (A) and the three-dimensional side view (B) generated by the protein structure homology modeling program Swiss PDB. It is a structural model of the chimeric anti-CEACAM-1 antibody (CM-10) V region of the top view (A) and the three-dimensional side view (B) generated by the protein structure homology modeling program Swiss PDB. Coomassie blue-stained SDS-PAGE gel of protein A purified antibody. Approximately 2 μg of each sample was loaded onto a NuPAGE 4-12% Bis-Tris gel (Invitrogen catalog number NP0322BOX) and run at 200 V for 40 minutes. (A) IgG1 CDR-transplanted mutant; (B) IgG4 (S241P) CDR-transplanted mutant. Pageler Plus (SM1811) from Fermentas was used as the molecular weight standard (containing reference bands of 10, 25, and 70 kDa). The samples were numbered as follows:

It is a graph of the result of the competitive binding ELISA assay of recombinant human CEACAM-1. Various concentrations of purified humanized IgG antibody variants competed with constant concentrations of biotinylated anti-CEACAM-1 IgG (chimeric CM-10 IgG1): (A) IgG1 mutants; and (B) IgG4 (S241P). Mutant. Bound biotinylated chimeric CM-10 was detected using streptavidin HRP and TMB substrate. It represents the synergistic effect of anti-CEACAM1 and anti-PD-1 antibodies on the cytotoxicity of human TIL cells against human melanoma cells. TIL cells were subjected to humanized mAbs (black dashed lines, spherical markers) against human CEACAM1 at various concentrations, mAbs (gray solid lines, rectangular markers) against human PD-1, or a combination of both antibodies (black solid lines, triangles). Marker) and incubated. Melanoma cells treated with IFN-γ were added during the overnight incubation. The results represent the mean ± standard error of% cytotoxicity as determined by the classical LDH release assay from 3 wells per treatment. * P <0.05, paired T-test compared to monoclonal antibody against human CEACAM1 alone. Synergistic effect of anti-CEACAM1 and anti-PD-1 antibody on granzyme B level and cytotoxicity of human TIL cells on human melanoma cells when anti-PD-1 antibody was added before addition of anti-CEACAM1 antibody. Human melanoma cells were grown in the presence of IFN-α, which induces PD-L1 expression. Human TIL cells, medium only (black), non-specific IgG antibody (white), monoclonal antibody against human CEACAM1 at various concentrations (vertical line), monoclonal antibody against human PD-1 (horizontal line), or both antibodies Incubated with the combination (dots). (A) Results represent mean ± standard error of% cytotoxicity as determined by classical LDH release assay from 3 wells per treatment. * P ≤ 0.05, a paired T-test compared to a-PD-1 alone. (B) Results represent Granzyme B level ± standard error as determined by a commercially available Granzyme B ELISA kit from 3 wells per treatment. Synergistic effect of anti-CEACAM1 and anti-PD-1 antibody on granzyme B level and cytotoxicity of human TIL cells on human melanoma cells when anti-PD-1 antibody was added before addition of anti-CEACAM1 antibody. Human melanoma cells were grown in the presence of IFN-α, which induces PD-L1 expression. Human TIL cells, medium only (black), non-specific IgG antibody (white), monoclonal antibody against human CEACAM1 at various concentrations (vertical line), monoclonal antibody against human PD-1 (horizontal line), or both antibodies Incubated with the combination (dots). (A) Results represent mean ± standard error of% cytotoxicity as determined by classical LDH release assay from triple wells per treatment. * P ≤ 0.05, a paired T-test compared to a-PD-1 alone. (B) Results represent Granzyme B level ± standard error as determined by a commercially available Granzyme B ELISA kit from 3 wells per treatment. Synergistic effect of anti-CEACAM1 and anti-PD-1 antibody on the cytotoxicity of human LAK cells against human melanoma cells when the anti-PD-1 antibody is added before the addition of the anti-CEACAM1 antibody. Human melanoma cells were grown in the presence of IFN-α, which induces PD-L1 expression. Human LAK cells produced by activation of PBMC with IL-2 from healthy human donors, medium only (white), non-specific IgG antibody (black), monoclonal antibody against human CEACAM1 at various concentrations (vertical line) ), Monoclonal antibody against human PD-L1 (horizontal line), or a combination of both antibodies (dot). The results represent the mean ± standard error of% cytotoxicity as determined by the classical LDH release assay from 3 wells per treatment. * P ≤ 0.05, a paired T-test compared to a-PD-L1 alone. The combination index was calculated as described above. Treatment with anti-CEACAM1 antibody increases PD-L1 expression on target cancer cells. NK cells (NK92MI) were incubated with and without CM-24 (10 μg / ml) and then human melanoma cells (SKMEL28) were added. These cells were incubated for 24 hours, 48 hours and 72 hours and PD-L1 levels were measured at each time point by FACS analysis. Mean ratio of anti-PD-L1 compared to isotype controls suitable for the treatments shown at different time points. Synergistic effect of anti-CEACAM1 and anti-PD-1 antibodies on tumor progression in immune-competent mice. Mouse lymphoma cells were subcutaneously transplanted into the abdomen of BALB / C mice (day 1). On days 10, 15 and 20, mice were given PBS (black dashed line, white circle), anti-mouse CEACAM1 antibody (gray solid line, gray rectangle), anti-mouse PD-1 antibody (gray solid line, gray triangle). ) Or a combination of both antibodies (solid black line, black sphere) was administered intravenously. The anti-CEACAM1 antibody increases the cytotoxicity of human LAK cells against human melanoma cells. Human LAK cells were incubated with different concentrations of CM-24 for 30 minutes at 37 ° C. Human melanoma cancer cells (SKMEL28) were added for a 24-hour incubation. The results represent the mean ± standard error of% cytotoxicity as determined by the classical LDH release assay from 3 wells per treatment. * P ≤ 0.05, paired T-test compared to effector + target cells with medium only. The anti-CEACAM1 antibody increases the cytotoxicity of human LAK cells against human pancreatic cancer cells T3M4. Human LAK cells were incubated with different concentrations of CM-24 for 30 minutes at 37 ° C. Human pancreatic cancer cells T3M4 were added for a 24-hour incubation. The results represent the mean ± standard error of% cytotoxicity as determined by the classical LDH release assay from 3 wells per treatment. * P <0.05, paired T-test compared to effector + target cells with medium only. The anti-CEACAM1 antibody enhances IFN-γ secretion in human LAK cells in the presence of human cancer cells. Human LAK cells were incubated with different concentrations of CM-24 for 30 minutes at 37 ° C. Human lung cancer cells H358 (11A) or H460 (11B) were added during a 24-hour incubation. IFN-γ secretion was measured by Elisa. The results represent the mean + standard error of the emission values of Granzyme B for 3 iterations per treatment. * P <0.05, paired T-test compared to effector + target cells with medium only. The anti-CEACAM1 antibody enhances IFN-γ secretion in human LAK cells in the presence of human cancer cells. Human LAK cells were incubated with different concentrations of CM-24 for 30 minutes at 37 ° C. Human lung cancer cells H358 (11A) or H460 (11B) were added during a 24-hour incubation. IFN-γ secretion was measured by Elisa. The results represent the mean + standard error of the emission values of Granzyme B for 3 iterations per treatment. * P <0.05, paired T-test compared to effector + target cells with medium only. It shows the correlation between the expression of CEACAM1 type CEACAM1-long (A) and CEACAM1-short (B) in cancer cells, and resistance to inhibitors of B-Raf mutants. It shows the correlation between the expression of CEACAM1 type CEACAM1-long (A) and CEACAM1-short (B) in cancer cells, and resistance to inhibitors of B-Raf mutants. FIG. 13A shows a pictogram of lungs removed from mice transplanted with melanoma cells and treated according to the treatment group shown in the figure; FIG. 13B shows the average tumor weight of each treatment group; FIG. 13C shows the average tumor weight. , The number of lesions per mouse in each treatment group is shown. FIG. 13A shows a pictogram of lungs removed from mice transplanted with melanoma cells and treated according to the treatment group shown in the figure; FIG. 13B shows the average tumor weight of each treatment group; FIG. 13C shows the average tumor weight. , The number of lesions per mouse in each treatment group is shown. FIG. 13A shows a pictogram of lungs removed from mice transplanted with melanoma cells and treated according to the treatment group shown in the figure; FIG. 13B shows the average tumor weight of each treatment group; FIG. 13C shows the average tumor weight. , The number of lesions per mouse in each treatment group is shown. FIG. 6 is a bar histogram showing the percentage of CEACAM1 receptor occupancy in TIL cells isolated from a lung lesion model. 15A and 15B represent the light and heavy chain amino acid sequences of the reverted humanized anti-CEACAM1 mAb, named CM-24, which is currently in clinical trials. In FIG. 15A, the light chain sequence (SEQ ID NO: 52) is contained, amino acid residues 1 to 107 are variable regions containing CDRs, and amino acid residues 108 to 214 (bold) are κ light chain constant regions. FIG. 15B represents the heavy chain sequence (SEQ ID NO: 53), where amino acid residues 1-120 are variable regions and amino acid residues 121-447 (bold) are IgG4 heavy chain constant regions, predicted N-. The glycosylation site (asparagine 297) is underlined. FIG. 15C represents the heavy chain sequence (SEQ ID NO: 59), where amino acid residues 1-121 are variable regions and amino acid residues 122-450 (bold) are IgG1 heavy chain constant regions, predicted N-. The glycosylation site (asparagine 300) is underlined. 15A and 15B represent the light and heavy chain amino acid sequences of the reverted humanized anti-CEACAM1 mAb, named CM-24, which is currently in clinical trials. In FIG. 15A, the light chain sequence (SEQ ID NO: 52) is contained, amino acid residues 1 to 107 are variable regions containing CDRs, and amino acid residues 108 to 214 (bold) are κ light chain constant regions. FIG. 15B represents the heavy chain sequence (SEQ ID NO: 53), where amino acid residues 1-120 are variable regions and amino acid residues 121-447 (bold) are IgG4 heavy chain constant regions, predicted N-. The glycosylation site (asparagine 297) is underlined. FIG. 15C represents the heavy chain sequence (SEQ ID NO: 59), where amino acid residues 1-121 are variable regions and amino acid residues 122-450 (bold) are IgG1 heavy chain constant regions, predicted N-. The glycosylation site (asparagine 300) is underlined. 15A and 15B represent the light and heavy chain amino acid sequences of the reverted humanized anti-CEACAM1 mAb, named CM-24, which is currently in clinical trials. In FIG. 15A, the light chain sequence (SEQ ID NO: 52) is contained, amino acid residues 1 to 107 are variable regions containing CDRs, and amino acid residues 108 to 214 (bold) are κ light chain constant regions. FIG. 15B represents the heavy chain sequence (SEQ ID NO: 53), where amino acid residues 1-120 are variable regions and amino acid residues 121-447 (bold) are IgG4 heavy chain constant regions, predicted N-. The glycosylation site (asparagine 297) is underlined. FIG. 15C represents the heavy chain sequence (SEQ ID NO: 59), where amino acid residues 1-121 are variable regions and amino acid residues 122-450 (bold) are IgG1 heavy chain constant regions, predicted N-. The glycosylation site (asparagine 300) is underlined.

The present invention discloses a non-fully humanized monoclonal antibody that recognizes CEACAM1. Advantageously, the antibodies of the invention are almost completely humanized, thus avoiding the risk of adverse immune responses to the antibodies and are therefore safe for in vivo use in humans. The antibodies of the invention are characterized by having a unique CDR sequence as well as a novel non-fully humanized framework sequence and design. The unique properties of the monoclonal antibodies of the invention extend their therapeutic utility in the treatment and diagnosis of additional types of malignancies and various infectious diseases. More specifically, the monoclonal antibodies provided by the present invention have a particular combination of CDRs and non-fully humanized framework sequences, with unique properties over known non-human anti-CEACAM1 antibodies as well as improved safety. Has sex and potency.

The unique properties of the antibodies provided by the present invention require long-term or repeated doses, especially when no other non-human antibody can be administered due to the risk of inducing an immunogenic response to the non-human antibody itself. It provides some advantages to the use of these antibodies in humans. It becomes more important to avoid such an immune response when the person being treated is a patient suffering from a disease for which further deterioration of the patient's health should be avoided. The present invention further comprises, in another embodiment, (i) CDR1, CDR2 and CDR3 comprising the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the amino acids set forth in SEQ ID NO: 9. A heavy chain variable region containing a non-CDR (framework) amino acid sequence in which the sequence differs from 2 to 9 amino acids; and / or (ii) contains the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. A non-fully humanized monoclonal antibody comprising a light chain variable region containing CDR1, CDR2 and CDR3 and containing a non-CDR amino acid sequence containing 2-4 amino acids different from the amino acid sequence set forth in SEQ ID NO: 13; and specificizing human CEACAM1. Provided are analogs, derivatives and antigen-binding fragments thereof that are specifically recognized.

For clarity, the variable regions of the antibody provided by the present invention are also named (a) the CDR sequences previously described by the inventors of the present invention and (b) the non-CDR sequences herein. It should be emphasized that at least one of the framework sequences differs from the corresponding fully human framework sequence by at least one residue.

In certain embodiments, the phrase "sequence different from another sequence" as used herein refers to at least one amino acid substitution, at least one amino acid insertion, at least one relative to each sequence. Means a sequence containing a deletion of an amino acid, or any combination thereof. In certain embodiments, the phrase "sequence different from another sequence" as used herein means a sequence containing at least one amino acid substitution as compared to each sequence. As used herein, the term "non-CDR sequence" refers to a framework sequence that is not the CDR sequence identified by the present invention, i.e., any amino acid sequence contained within the variable region of an antibody. Examples of non-CDR sequences include sequences preceding or adjacent to CDR1, sequences between CDR1 and CDR2, sequences between CDR2 and CDR3, and sequences following or adjacent to CDR3.

Since the variable regions of the antibodies provided by the present invention differ from the variable regions of fully human antibodies by at least one amino acid, they are also labeled as "incompletely humanized" and "incompletely human" antibodies.

The term "CEACAM1" is used to refer to a protein product of the CEACAM1 gene, such as NP_001020083.1, NP_001703.2. In humans, 11 different CEACAM1 splice variants have been detected so far. Each CEACAM1 isoform has a number of extracellular immunoglobulin-like domains (eg, CEACAM1 with four extracellular immunoglobulin-like domains is known as CEACAM1-4), membrane scaffolds and / or the length of their cytoplasmic tail. (For example, CEACAM 1-4 with a long cytoplasmic tail is known as CEACAM 1-4L, CEACAM 1-4 with a short cytoplasmic tail is known as CEACAM 1-4S). The N-terminal domain of CEACAM1 begins immediately after the signal peptide and its structure is considered to be IgV type. For example, in CEACAM1 commentary P13688, the N-terminal IgV-type domain is composed of 108 amino acids at amino acids 35-142. This domain was identified as involved in allogeneic affinity binding activity (Watt et al., 2001, Blood. 98, 1469-79). All variants, including these splicing variants, are included within the term "CEACAM1".

The terms "anti-CEACAM1 antibody", "antibody that recognizes CEACAM1", "antibody against CEACAM1" and "antibody against CEACAM1 (antibody to CEACAM1)" are interchangeable and have sufficient affinity and specificity. As used herein to refer to an antibody that binds to the CEACAM1 protein.

As used herein, the term "antigen" refers to a molecule or portion of a molecule that induces antibody formation and to which the antibody can bind. The antigen can have one or more epitopes. The specific reaction mentioned above means that the antigen reacts with its corresponding antibody in a highly selective manner and does not react with many other antibodies that can be induced by other antigens. .. The antigen of the present invention is the CEACAM1 protein or a fragment thereof.

As used herein, the term "antigen determinant" or "epitope" refers to a region of an antigen molecule that specifically reacts with a particular antibody. Epitope-derived peptide sequences can be used alone or in combination with carrier moieties to immunize animals and produce additional polyclonal or monoclonal antibodies, applying methods known in the art. it can. The isolated peptide derived from the epitope can be used in a diagnostic method for detecting an antibody, and can be used as a therapeutic agent when inhibition of the antibody is required.

The antibody or immunoglobulin contains two heavy chains and two light chains that are linked together by a disulfide bond, and each light chain is linked to each heavy chain by a disulfide bond in a "Y" shape. Digestion by proteolysis of antibodies produces FV (Fragment variable (variable fragment)) and FC (Fragment crystallinity (crystalline fragment)) domains. The Fab of the antigen-binding domain contains a region in which the polypeptide sequence changes. The term F (ab') ₂ refers to _two Fab'arms linked together by a disulfide bond. The central axis of the antibody is called the Fc fragment. Each heavy chain has a variable domain ( _VH ) at one end, followed by several constant domains ( _CH ). Each light chain has a constant domain (C _L) at variable domain (V _L) and the other end to one end, the light chain variable domain is aligned in the variable domain and one row of the heavy chain, light chain constant domain first It is aligned with the constant domain (CH1) of the heavy chain of 1. The variable domains of each pair of light and heavy chains form an antigen-binding site. The domains on the light and heavy chains have the same general structure, and each domain is relatively conserved in sequence and is linked by three hypervariable domains known as complementarity determining regions (CDRs 1-3). Includes four framework regions. These domains contribute to the specificity and affinity of the antigen binding site. The heavy chain isotype (gamma, alpha, delta, epsilon or mu) determines the immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively). The light chain is one of two isotypes found in all antibody classes (kappa, kappa or lambda, λ).

The term "antibody" is used in the broadest sense, including monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, polyvalent antibodies, multispecific antibodies (eg, bispecific antibodies), and Includes an antibody fragment long enough to exhibit the desired biological activity.

The antibody of the present invention is a molecule containing at least an antigen-binding portion of the antibody. One antibody or plurality of antibodies of the present invention comprises an intact antibody such as a polyclonal antibody or a monoclonal antibody (mAb) and a proteolytic fragment thereof such as a Fab or F (ab') ₂ fragment. Single chain antibodies also fall within the scope of the invention.

An "antibody fragment" generally comprises the antigen binding site of an intact antibody and thus contains only a portion of the intact antibody that retains the ability to bind the antigen. Examples of antibody fragments included by the definition of the present invention are (i) Fab fragments with VL, CL, VH and CH1 domains; (ii) Fab fragments with one or more cysteine residues at the C-terminus of the CH1 domain. Fab'fragment; (iii) Fd fragment having VH and CH1 domains; (iv) Fd'fragment having VH domain and CH1 domain and one or more cysteine residues at the C-terminus of CH1 domain; (v) antibody Fv fragment with single arm VL and VH domains; (vi) dAb fragment consisting of VH domain (Ward et al., Nature 1989, 341, 544-546); (vi) isolated CDR region; viii) 'which is a bivalent fragment comprising a fragment F (ab' 2 two Fab linked by a disulfide bridge at the hinge _{region) 2} fragments; (ix) single chain antibody molecules (e.g., single chain Fv; scFv) ( Bird et al., Science 1988,242,423-426; and Huston et al., PNAS (USA) 1988,85,5879-5883); (x) in the light chain variable domain (VL) within the same polypeptide chain. A "diabody" having two antigen binding sites, including linked heavy chain variable domains (VHs) (eg, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448); (xi) A pair of tandem Fd segments (VH-CH1-VH-) forming a pair of antigen-binding regions with complementary light chain polypeptides. "Linear antibodies" containing CH1) (Zapata et al. Protein Eng., 1995, 8, 1057-1062; and US Pat. No. 5,641,870).

Single chain antibodies are single chain complex polypeptides that have the ability to bind antigen and contain amino acid sequences that are homologous or similar to the variable regions of the immunoglobulin light and heavy chains, i.e., linked _VH- V. It may be _L or single chain _{F V (SC F} V).

As used herein, the term "neutralizing antibody" can reduce or inhibit (block) signal transduction via activity or receptors, as determined by in vivo or in vitro assays according to specifications. Refers to a molecule that has an antigen-binding site for a specific receptor or ligand target.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population have the potential for natural mutations that may be present in small amounts. It is the same except. Monoclonal antibodies are highly specific and have been made against a single antigen. Moreover, in contrast to polyclonal antibody preparations containing different antibodies typically made against different determinants (epitopes), each monoclonal antibody is made against a single determinant on the antigen. There is. The modifier "monoclonal" should not be construed as requiring the production of antibodies by any particular method. Monoclonal Abs can be obtained by methods known to those of skill in the art. For example, monoclonal antibodies used in accordance with the present invention are first described in Kohler et al. , Nature 1975, 256,495, or by the recombinant DNA method (see, eg, US Pat. No. 4,816,567). "Monoclonal antibodies" are also described, for example, by Clackson et al. , Nature 991, 352, 624-628 or Marks et al. , J. Mol. Biol. , 1991,222: 581-597, may be isolated from the phage antibody library.

The mAbs of the present invention can be of any immunoglobulin class, including IgG, IgM, IgE, or IgA. Hybridomas that produce mAbs can be cultured in vitro or in vivo. High titers of mAbs can be obtained by in vivo production and when cells from individual hybridomas are injected intraperitoneally into pristine-primed Balb / c mice, ascites containing high concentrations of the desired mAbs. To produce. Monoclonal Abs of isotype IgM or IgG can be purified from such ascites or from culture supernatants using column chromatography methods well known to those of skill in the art.

As used herein, the term "human antibody" is any of the techniques for producing human antibodies that have an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or that are disclosed herein. Refers to an antibody produced using This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues. Human antibodies can be produced using a variety of techniques known in the art.

As used herein, the terms "molecule with antigen-binding portion of an antibody" and "antigen-binding fragment" are used not only for any isotype of intact immunoglobulin molecule produced by any animal cell line or microorganism. Fab fragment, Fab'fragment, F (ab') ₂ fragment, heavy and / or variable portion of light chain thereof, Fab mini-antibody (the entire contents of which are incorporated herein by reference, WO 93/15210, USA). See patent application 08 / 256,790, WO 96/13583, US patent application 08 / 817,788, WO 96/37621, US patent application 08/999,554), dimer bispecific mini. A single-stranded antibody comprising, but not limited to, an antigen-binding reactive portion thereof as well as incorporating such a reactive portion, and such antibody-reactive moiety are physically inserted, including but not limited to antibodies (see Muller et al., 1998). It is intended to include any other type of molecule. Such molecules may be provided by any known technique including, but not limited to, enzymatic cleavage, peptide synthesis or recombination techniques.

The present invention also provides conservative amino acid variants of the antibody molecules of the present invention. Variants of the invention that preserve the overall molecular structure of the coding protein can also be made. Given the properties of the individual amino acids, including the disclosed protein products, some reasonable substitutions will be recognized by those of skill in the art. Amino acid substitutions, or "conservative substitutions," can be made, for example, based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues involved. As used herein, the term "antibody analog" refers to an antibody derived from another antibody with one or more conservative amino acid substitutions.

As used herein, the term "antibody variant" refers to any molecule, including the antibodies of the invention. For example, a fusion protein in which an antibody or antigen-binding fragment thereof is linked to another chemical entity is considered an antibody variant.

As used herein, the term "incompletely humanized monoclonal antibody" refers to any amino acid sequence flanking or / or immediately adjacent to the CDR that is not fully human, i.e. taken from a native human antibody. Refers to a monoclonal antibody having a heavy chain and / or a light chain variable domain that is not identical to a known homologous sequence or corresponding sequence of.

In pharmaceuticals and pharmaceutical formulations, the activator is preferably utilized with one or more pharmaceutically acceptable carriers (s) and, optionally, any other therapeutic ingredient. The carrier (s) must be pharmaceutically acceptable in the sense that it is compatible with the other ingredients of the formulation and is not overly harmful to its recipient. The activator is provided in an amount effective to achieve the desired pharmacological effect and in an amount appropriate to achieve the desired daily dose, as described above.

Typically, the molecules of the invention that contain an antigen-binding portion of an antibody or that contain another polypeptide that contains a peptide mimetic are suspended in sterile saline for therapeutic use. The pharmaceutical composition may optionally be formulated to control the release of the active ingredient (the molecule containing the antigen-binding portion of the antibody) or to prolong its presence in the patient's system. A number of suitable drug delivery systems are known, including, for example, implantable drug release systems, hydrogels, hydroxymethyl celluloses, microcapsules, liposomes, microemulsions, and microspheres. Controlled release formulations can be prepared through the use of polymers in the complex or can adsorb the molecules of the invention. For example, biocompatible polymers include a matrix of poly (ethylene-co-vinyl acetate) and a matrix of polyanhydride copolymers of stearic acid dimer and sebacic acid. The rate of release of a molecule of the invention, an antibody or antibody fragment, from such a matrix depends on the molecular weight of the molecule, the amount of molecules in the matrix, and the size of the dispersed particles.

The pharmaceutical composition of the present invention is any suitable such as oral administration, local administration, intranasal administration, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intra-articular administration, intralesional administration, parenteral administration and the like. Can be administered by means. Intravenous (iv) administration is usually used.

The therapeutically effective amounts of the molecules of the invention are, among other things, the dosing schedule, the unit dose of the molecule to be administered, whether the molecule is administered in combination with other therapeutic agents, the patient's immune status and health status, and the administration. It is clear to those skilled in the art that it depends on the therapeutic activity of the molecule and the judgment of the treating physician. As used herein, "therapeutically effective amount" refers to the amount of molecule required to alleviate one or more symptoms associated with a disorder being treated over a period of time.

The term "therapeutically effective amount" refers to the amount of drug effective in treating a disease or disorder in a mammal. In the case of cancer, therapeutically effective doses of the drug reduce the number of cancer cells; reduce tumor size; inhibit the infiltration of cancer cells into peripheral organs (ie, slow down to some extent, preferably stop); tumors It inhibits metastasis (ie, slows it down to some extent, preferably stops it); inhibits tumor growth to some extent; and / or can alleviate one or more of the symptoms associated with the disorder to some extent. The drug can be cytostatic and / or cytotoxic to the extent that the drug can prevent growth and / or kill existing cancer cells. For cancer therapy, in vivo efficacy can be measured, for example, by assessing survival, time to disease progression (TTP), kinetics (RR), duration of response, and / or quality of life. it can.

The term "sugar" refers to monosaccharides, disaccharides, and polysaccharides. Examples of sugars include, but are not limited to, sucrose, trehalose, dextrose and the like.

As is well known, the molecules of the present invention as active ingredients are dissolved, dispersed or mixed in excipients that are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol and the like and combinations thereof. Other suitable carriers are well known to those of skill in the art. In addition, if desired, the composition can contain small amounts of auxiliary substances such as wetting agents or emulsifiers, pH buffers.

The pharmaceutical composition of the present invention may be administered together with the antitumor composition. According to a specific embodiment, the antitumor composition comprises at least one chemotherapeutic agent. Chemotherapeutic agents that can be administered with or separately from the antibodies of the invention include, but are not limited to, any such agent known in the art that exhibits anticancer activity. May include: mitoxanthrone, topoisomerase inhibitor, spindle poison binka: vincristine, vincristine, binorerbin (taxol), paclitaxel, docetaxel; alkylating agents: mechloretamine, chlorambusyl, cyclophosphamide, merphalan, ifosphamide; methotrexate; 6-Mercaptopurine; 5-fluorouracil, citarabin, gemcitabine; podophyllotoxin: etopocid, irinotecan, topotecan, dacarbadine; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin; nitrosourea: carmustin (BCNU), romustine, epirubicin Idalubicin, daunorubicin; inorganic ions: cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxyphene, leuprolide, flutamide, and megestol acetate.

According to certain embodiments, the chemotherapeutic agent is an alkylating agent, an antimetabolite, a folic acid analog, a pyrimidine analog, a purine analog and related inhibitors, a binca alkaloid, an epipodophylrotoxin, an antibiotic, L-sparaginase, topoisomerase inhibitor, interferon, platinum coordination complex, anthracendion-substituted urea, methylhydrazine derivative, corticosuppressant, adrenocorticosteroids, progestins, estrogens, anti-estrogens, androgens, anti-androgens, And selected from the group consisting of gonadotropin-releasing androgen analogs. According to another embodiment, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel and doxetaxel. Two or more chemotherapeutic agents can be used in cocktails for administration in combination with administration of anti-CEACAM1 antibody.

As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or prophylactic measures. Those in need of treatment include those who already have a disability and those whose disability should be prevented.

The terms "cancer" and "cancerous" refer to or describe physiological conditions in mammals that are typically characterized by disordered cell proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include sarcoma, lung cancer, thyroid cancer, breast cancer, colon cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, cervical cancer, pancreatic cancer, leukemia, lymphoma, bone marrow cancer, ovary. Examples include cancer, uterine cancer, sarcoma, bladder cancer, or endometrial cancer.

The term "antitumor composition" is useful in the treatment of cancer, comprising at least one active therapeutic agent capable of inhibiting or preventing tumor growth or function and / or causing destruction of tumor cells. Refers to a composition. Suitable therapeutic agents for treating cancer in antitumor compositions target cytokines such as chemotherapeutic agents, radioisotopes, toxins, interferons, and cytokines, cytokine receptors or antigens associated with tumor cells. Includes, but is not limited to, antagonists. Preferably, the therapeutic agent is a chemotherapeutic agent.

As used herein, the term "diagnosing" refers to determining the presence or absence of a medical condition, classifying a medical condition or symptom, determining the severity of a medical condition, monitoring the progression of the medical condition, and the outcome of the medical condition. And / or to predict the outlook for recovery.

As used herein, the term "mutation of an amino acid residue" refers to the substitution, insertion, or deletion of a single amino acid residue. As used herein, the term "return mutation of an amino acid residue" refers to the substitution of a single amino acid residue found in the human antibody framework for the corresponding amino acid residue found in the mouse antibody framework.

The following examples are presented to more fully illustrate some embodiments of the invention. However, they should by no means be construed as limiting the broad scope of the invention.

Example

The reference fully humanized heavy chain sequence (SEQ ID NO: 57) into which the reversion mutation is introduced consists of:
QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO: 7) -GYAFTNNLIE (SEQ ID NO: 1) -WVRQAPGQGLEWMG (SEQ ID NO: 8) -VINPGSGDTNYNEKFKG (SEQ ID NO: 2) -RVTMTRDTSISTAYM

The reference fully humanized light chain sequence (SEQ ID NO: 58) into which the reversion is introduced consists of:
DIQMTQSLSLSVSVGDRVTITC (SEQ ID NO: 11) -RTSQDIGNYLN (SEQ ID NO: 4) -WYQQKPGKAPKLLIY (SEQ ID NO: 12) -YTSRLHS (SEQ ID NO: 5) -GVPSRFSGSGSGTDFTFSSLQPEDITYC (SEQ ID NO: 13)

Example 1. Design of CDR Transplanted Antibody Variable Region Sequences To identify amino acids in the V region framework that may be important for antibody binding properties, a structural model of the chimeric anti-CEACAM-1 antibody V region was generated using Swiss PDB. , Analyzed (Fig. 1). These amino acids were found for integration into one or more mutant CDR transplanted antibodies. Both the V _H and V _κ sequences of CM-10 contain typical framework residues, and the motifs of CDR1, 2 and 3 are comparable to many mouse antibodies. These CDRs were obtained directly from the mouse sequence. The Swiss PDB model was then analyzed with mouse / human homology at critical positions to highlight framework regions and individual residues that could potentially affect the presentation of CDRs.

Mutant Design To identify heavy and light chain human sequences with the highest homology for use as a V-region framework, heavy and light chain V-region amino acid sequences of human germline V-region sequences. Compared to the database. By transplanting the CDRs onto the framework, if necessary, a series of reversions to the mouse sequence of the residues identified above that are potentially important for the recovery of chimeric antibody binding efficiency. Heavy and light chain V regions were designed. It was considered that a small number of mouse residues needed to be retained in each mutant. Potential T cell epitopes are then determined by the application of Antitope's insilico technology iTope ™ and TCED ™ (T cell epitope database) (Perry et al 2008, Bryson et al 2010). The mutant sequence with the lowest incidence was selected. The number of reversion mutations was determined by the starting mouse sequence. From the structural analysis, up to 13 sites were identified in V _H and 5 sites in V _κ were identified as potentially structurally important residues. We prioritized these and designed mutants to incorporate them in varying numbers. There is no theoretical limit to the number of reversion mutations, but the more reversion mutations that are incorporated, the fewer human sequences can be. Five V _H chains and three V _κ chains were designed using the sequences set forth in SEQ ID NOs: 28-35.

iTope ™ and TCED ™
The iTope ™ software provides that within the binding groove of 34 human MHC class II alleles, between the amino acid side chains of the peptide and specific binding pockets (particularly pocket positions; p1, p4, p6, p7 and p9). Predict good interactions. The location of key binding residues is achieved by in silico production of 9-mer peptides that are duplicated by a single amino acid across the test protein sequence. In-house comparisons with physical MHC class II binding experiments have shown that iTope ™ can be used to adequately distinguish between peptides that bind to MHC class II molecules and those that do not. .. However, these results indicate that all methods of predicting MHC class II binding allow other important processes during antigen presentation, such as protein / peptide processing, recognition by T cell receptors or T cell tolerance to peptides. Therefore, it should be evaluated in the light of the fact that the number of T cell epitopes is essentially over-predicted.

TCED ™ contains all peptide sequences previously screened in the EpiScreen ™ T cell epitope mapping assay. TCED ™ is an optional test against a large (> 10,000 peptide) database of peptides by BLAST search to specifically select segments previously shown not to stimulate T cell response. Used to search for arrays. In addition, any region in the database that has significant homology with the T cell epitope was excluded.

Construction of CDR-Transplanted Mutants The genes for the CDR-transplanted V _H region and V _κ region of all mutants of CM-10 are synthesized by annealing, ligation, and PCR amplification using a series of overlapping oligonucleotides, and complete. A long synthetic V region was obtained. Then, a mutant was constructed and cloned directly into an expression vector system for the _{V H} chain and V _kappa chain both IgG1 and IgG4 (S241P). All constructs were confirmed by sequencing.

Construction of an antibody, all combinations of expression and purification chimeric genes and CDR grafted _{V H} chain and V _kappa chain (i.e., a total of 15 pairs for each of the IgG1 and IgG4 (S241P)), HEK293- EBNA (ATCC Cat. No. CRL-10852) Cells were transiently transfected with calcium phosphate. Transfections were incubated for up to 5 days prior to supernatant collection.

The chimeric antibody and the CDR-transplanted variant of CM-10 were purified from the transient cell culture supernatant on a Protein A Sepharose column (GE Healthcare Catalog No. 1100034-93) and buffered to 1 × PBS pH 7.4. Exchanged and quantified by OD _{280 nm} using absorptiometry based on the predicted amino acid sequence (CM-10 chimeric antibody and variant EC _(0.1%) = about 1.37 to 1.40). .. Chimeric antibodies and major humanized variants were analyzed by reducing SDS-PAGE. Bands corresponding to the expected sizes of the _VH and _Vκ chains were found, with no evidence of any contamination (Fig. 2).

Example 2. Competitive binding of purified antibody to human CEACAM-1 Binding of the purified chimeric CM-10 antibody to recombinant human CEACAM-1 along with each of the chimeric antibody and the CDR-transplanted mutant was evaluated by competitive ELISA. A dilution series (3-fold) of 20 μg / ml to 0.009 μg / ml of chimeric or humanized antibody was premixed with a constant concentration of biotinylated chimeric CM-10 (0.005 μg / ml, final concentration), followed by 1 × Nunc Immuno MaxiSorp 96-well flat-bottomed microtiter plate precoated with 0.5 μg / ml recombinant human CEACAM-1 (R & D Systems Catalog No. 2244-CM-050) diluted at PBS pH 7.4 (Fisher Catalog No. DIS) -971-030J) was incubated at room temperature for 1 hour. Binding of biotinylated antibody was detected on streptavidin-HRP (Sigma Catalog No. S5512) and TMB substrate (Invitrogen Catalog No. 00-2023). The reaction was stopped with 3M HCl and the absorbance was read at 450 nm on a Dynex Technologies MRX TC II plate reader and the binding curve was plotted.

All humanized variants provided a binding profile to chimeric CM-10 similar to the binding curve shown in FIG. Using these data, the IC ₅₀ values of each antibody were calculated and normalized to the IC _{50 of the} chimeric CM-10 contained in each ELISA and shown in Tables 6 and 7.

_{IC 50} data normalized for all mutants tested showed a range of 0.84 to 1.56, the coupling efficiency of all of the CDR-grafted antibodies against human CEACAM-1 is a chimeric CM-10 coupling efficiency equivalent It was shown that.

Example 3. Combination of humanized mAb against CEACAM1 and anti-PD-1 / PD-L antibody A. The synergistic effect of anti-CEACAM1 and anti-PD-1 antibody on the cytotoxicity of human TIL cells on human melanoma cells was demonstrated. Human melanoma cancer cells (MALME 3M) were grown in the presence of IFN-α to induce PD-L1 expression. Human TIL cells (TIL14) are cloned into human CEACAM1 (CM-24) (0.01 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.5 μg / ml) and monoclonal antibody against human PD-1. (Clone E12.2H7) or a combination of both antibodies (each antibody 0.005, 0.025, 0.05 and 0.25 μg / ml) was incubated at 37 ° C. for 30 minutes. Prior to cytotoxicity assessment, IFN-α treated human melanoma cancer cells were added for an overnight incubation. FIG. 4 shows that both anti-CEACAM1 and anti-PD-1 antibodies were able to bind to their respective targets on human lymphocytes, such as TIL cells, and that this binding was to human cancer cells. It is shown that the toxicity of the drug was significantly enhanced over each monotherapy alone. Therefore, we conclude that protecting lymphocytes from immunosuppressive signals from target cancer cells results in substantial cytotoxicity to these cancer cells.

B. The synergistic effect of anti-CEACAM1 and anti-PD-1 antibody on the cytotoxicity of human TIL cells against Granzyme B level and human melanoma cells when the anti-PD-1 antibody was added before the addition of the anti-CEACAM1 antibody was also shown. : Human melanoma cancer cells (MALME 3M) were grown in the presence of IFN-α to induce PD-L1 expression. Human TIL cells (TIL14), medium only (black), non-specific IgG antibody (0.8 μg / ml, white), various concentrations (0.05 μg / ml, 0.1 μg / ml, 0.2 μg / ml) , 0.4 μg / ml, 0.8 μg / ml) CM-24, mAb against human PD-1 (clone E12.2H7) or a combination of both antibodies (0.05 μg / ml, respectively, 0.1 μg / ml, respectively) , 0.2 μg / ml respectively, 0.4 μg / ml respectively, 0.8 μg / ml respectively). A monoclonal antibody against human PD-1 was first added at 37 ° C. for 30 minutes, followed by mAb against human CEACAM1. Prior to cytotoxicity assessment, IFN-α treated human melanoma cancer cells were added for an overnight incubation. The combination index (CI) was calculated to be 0.15. In the same assay, the level of the cytotoxic protein Granzyme B secreted upon activation of cytotoxic cells was evaluated with a commercially available Granzyme B ELISA kit. FIG. 5 shows that anti-CEACAM1 and anti-PD-1 antibodies are capable of binding to their respective targets on human lymphocytes such as TIL cells, and that this binding is to granzyme B secretion and human TIL cells to human cancer cells. Shows that it enhances toxicity. FIG. 5 again shows that protection of lymphocytes from immunosuppressive signals from target cancer cells results in substantial cytotoxicity to target cancer cells, indicating that timing can be an important factor in combination therapy. Suggest.

C. Synergistic effect of anti-CEACAM1 and anti-PD-L1 antibody on granzyme B level and cytotoxicity of human TIL cells against human melanoma cells when anti-PD-1 antibody was added prior to addition of anti-CEACAM1 antibody (data not shown) ). Human melanoma cells (MALME 3M) were grown in the presence of IFN-α to induce PD-L1 expression. Human TIL cells (TIL14), medium only (black), non-specific IgG antibody (0.8 μg / ml, white), various concentrations (0.05 μg / ml, 0.1 μg / ml, 0.2 μg / ml) , 0.4 μg / ml, 0.8 μg / ml) monoclonal antibody against human CEACAM1 (CM-24), monoclonal antibody against human PD-L1 (clone 29E.2A3), or a combination of both antibodies (0.05 μg / ml, respectively). Incubated with ml (0.1 μg / ml each, 0.2 μg / ml each, 0.4 μg / ml each, 0.8 μg / ml each). The anti-PD-L1 antibody was first added at 37 ° C. for 30 minutes, followed by the monoclonal antibody against human CEACAM1. Prior to cytotoxicity assessment, IFN-α treated human melanoma cancer cells were added for an overnight incubation. The combination index (CI) was calculated to be 0.67. The results represent the mean ± standard error of% cytotoxicity as determined by the classical LDH release assay from 3 wells per treatment. * P ≤ 0.05, a paired T-test compared to a-PD-L1 only. In the same assay, the level of the cytotoxic protein Granzyme B secreted upon activation of cytotoxic cells was evaluated with a commercially available Granzyme B ELISA kit. The results represent the average Granzyme B level from 3 wells per treatment. These results show that anti-CEACAM1 and anti-PD-L1 antibodies can bind to their respective targets on human lymphocytes (such as TIL cells) and human cancer cells (such as melanoma cells). Moreover, it is shown that this binding enhances granzyme B secretion and toxicity of human TIL cells to human cancer cells. Furthermore, blocking the interaction of PD-1 / PD-L1 and CEACAM1 / CEACAM1 can result in synergistic effects and protects lymphocytes from immunosuppressive signals of PD-1 ^lymphocytes / PD-ligand ^{cancer cells.} This demonstrated substantial cytotoxicity to these cancer cells, regardless of whether they were antigen-targeted PD-1, PD-L1 or PD-L2.

D. Synergistic effect of anti-CEACAM1 and anti-PD-1 antibody on the cytotoxicity of human LAK cells against human melanoma cells when the anti-PD-1 antibody is added before the addition of the anti-CEACAM1 antibody. Human melanoma cells (SKMEL28, CEACAM1 positive, PD-L1 positive) were grown in the presence of IFN-α to induce PD-L1 expression. Human LAK (phosphokine activation killer) cells prepared by activation of PBMC with IL-2 (500 units / ml) from a healthy human donor for 7 days in medium only (white), non-specific IgG antibody (0.8 μg) Monoclonal antibody against human CEACAM1 (CM-24), human PD-1 at various concentrations (0.1 μg / ml, 0.2 μg / ml, 0.4 μg / ml, 0.8 μg / ml) (/ ml, black) It was incubated with a monoclonal antibody against (Clone E12.2H7) or a combination of both antibodies (0.1 μg / ml, respectively, 0.2 μg / ml, respectively, 0.4 μg / ml, respectively, 0.8 μg / ml, respectively). A monoclonal antibody against human PD-1 was first added at 37 ° C. for 30 minutes, followed by a monoclonal antibody against human CEACAM1. Prior to cytotoxicity assessment, IFN-α treated human melanoma cells were added for a 24-hour incubation. FIG. 6 shows that anti-CEACAM1 and anti-PD-1 antibodies can bind to their respective targets on activated human lymphocytes such as LAK cells, and that this binding toxicizes human LAK cells to human cancer cells. Indicates to be enhanced. The combination index (CI) was calculated to be less than 0.8. FIG. 6 further shows that the binding of these antibodies to LAK cells is somewhat interrelated, justifying further study of the binding mechanism, and that this mechanism is present in various activated lymphocytes. Is shown.

E. Treatment with anti-CEACAM1 antibody increases the expression of PD-L1 on target cancer cells. Human NK cells (NK92MI) were incubated with or without a monoclonal antibody against human CEACAM1 (10 μg / ml CM-24), after which human melanoma cancer cells (SKMEL28) were added. These cells were incubated for 24 hours, 48 hours and 72 hours and PD-L1 levels were measured at each time point by FACS analysis. The mean ratio levels of anti-PD-L1 compared to appropriate isotype controls for the treatments shown at different time points are shown in FIG. 7 showing that the expression of CEACAM1 and PD-L1 on cancer cells is actually interrelated. Shown. The addition of anti-CEACAM1 antibody increases the expression of PD-L1 on living cancer cells and therefore provides additional support for combination therapy with both agents. Since the number of PD-L1 proteins on cancer cells remains relatively high and sensitizes these cells with anti-PD-1 / PD-L1 antibody treatment, anti-CEACAM1 antibody is administered first, followed by In addition, treating cancer by administering anti-PD-L1 and / or anti-PD-L2 antibodies can be beneficial, meaning that combination therapy can improve clinical outcomes. Administration of different antibodies at different times, rather than simultaneously, maximizes the cytotoxic effect of lymphocytes on cancer cells. Without being bound by any theory or mechanism, this finding may relate to another surprising finding of the invention in that treatment with anti-CEACAM1 antibody increases PD-L1 expression on target cancer cells. Temporarily, this supports the need for multiple antibodies to obtain improved efficacy for cytotoxic lymphocytes. Administration of anti-PD-1 antibody first blocks PD-1 molecules on lymphocytes, followed by administration of anti-CEACAM1 antibody, blocking CEACAM1 molecules on lymphocytes and / or target cancer cells, targeting cancer. It can be envisioned to increase the expression of PD-1 ligands on cells. However, since PD-1 molecules on lymphocytes are already blocked, increased expression levels of PD-1 ligands on target cancer cells allow lymphocytes to efficiently exert their full cytotoxic potential. Do not interfere.

F. Synergistic effect of anti-CEACAM1 and anti-PD-1 antibody on tumor progression in immunocompetent mice. Mouse lymphoma cells ^{(5 × 10 6, A20)} , and allograft into the abdomen of Balb / C mice by subcutaneous injection on day 1. On day 10, the mean tumor volume reached 45 mm ^3, mice were randomized into separate 4 groups (11-12 animals per group), PBS, CC-1 (anti-mouse CEACAM1 antibodies, 6 mg / kg) , PRM-1 (anti-mouse PD-1 antibody, 6 mg / kg) or a combination of CC-1 and PRM-1 (6 mg / kg, respectively) was administered intravenously. Treatment was repeated on days 15 and 20. The effect of the monoclonal antibody alone on human CEACAM1, the effect of the monoclonal antibody alone on human PD-1, and the effect of the combination of both antibodies on tumor growth inhibition were observed. An immune competent for this experiment that allows assessment of the biological activity of anti-mouse CEACAM1 and anti-mouse PD-1 antibodies in mice with an intact immune system and assesses the overall immune system response to mouse cancer cells. Baby / C mice were selected. Overall, this model simulates treatment in humans in which cancer patients receive a combination of anti-human CEACAM1 and anti-human PD-1 / PD-L1 / PD-L2 antibodies. Without being bound by any theory or mechanism, the combination of anti-CEACAM1 and anti-PD-1 / PD-L1 / PD-L2 antibodies prohibits cancer cells from evading the patient's immune system activation and cytotoxicity. Therefore, it is assumed to bring about a remarkable anti-cancer reaction. FIG. 8 shows that anti-CEACAM1 and anti-PD-1 / PD-L1 / PD-L2 antibodies were able to bind in vivo to their respective targets on tumor cells and / or immune cells, which were combined. It is shown that the binding significantly attenuates tumor progression compared to each monotherapy. Efficacy and use of anti-CEACAM1 and anti-PD-1 / PD-L1 / PD-L2 combinations within a significant volume of established tumors, mimicking the clinical setting in which patients with established tumors are being treated This result is very important as it also proves the possibility of.

Example 4. Combination treatment with humanized mAb for CEACAM1 and LAK cells.
A. Anti-CEACAM1 antibody increases the cytotoxicity of human LAK cells against human melanoma cells: PBMC cells are isolated from healthy donors and then activated with IL-2 (500 or 1000 units / ml) for 3 days. , Human LAK cell population was prepared. Human LAK cells were then incubated with 0.1 μg / ml, 0.5 μg / ml, 2.5 μg / ml, 5 μg / ml or 10 μg / ml anti-CEACAM1 antibody (CM-24) for 30 minutes at 37 ° C. .. Human melanoma cells (SKMEL28) were added for a 24-hour incubation, after which cytotoxicity was determined. FIG. 9 shows that human LAK cells are cytotoxic to human melanoma cancer cells by themselves (eg, compared to the two bars on the left), but the addition of anti-CEACAM1 antibody is associated with these human melanomas. It is shown that the cytotoxicity to cancer cells is significantly enhanced in a dose-dependent manner.

B. The anti-CEACAM1 antibody enhances the cytotoxicity of human LAK cells against a variety of human pancreatic and lung cancer cells. PBMC cells were isolated from healthy donors and then activated with IL-2 (500 units / ml) for 7 days to create a human LAK cell population. Human LAK cells were then incubated with 0.1 μg / ml-10 μg / ml anti-CEACAM1 antibody (CM-24) at 37 ° C. for 30 minutes as shown. Three different types of human pancreatic cancer cells T3M4, SU8686 and PANC2, and two different types of human lung cancer cells H358 and H460 were added for a 24-hour incubation. FIG. 10 shows that human LAK cells are themselves cytotoxic to human pancreatic T3M4 cancer cells, but the addition of anti-CEACAM1 antibody significantly enhances the cytotoxicity to these human cancer cells in a dose-dependent manner. Show that. Similar results were obtained for other pancreatic and lung cancer cells.

C. The anti-CEACAM1 antibody promotes granzyme B secretion of human LAK cells in the presence of human pancreatic and lung cancer cells. Human LAK cells were incubated with anti-CEACAM-1 antibody (CM-24) at different concentrations at 37 ° C. for 30 minutes. Human pancreatic cancer cells T3M4 (A) or human lung cancer cells H358 (B) were then added for a 24-hour incubation. Granzyme B secretion was measured by ELISA. The results show that human LAK cells produce high levels of granzyme B on their own, whereas the addition of anti-CEACAM1 antibody significantly increases granzyme B levels in a dose-dependent manner.

D. The anti-CEACAM1 antibody promotes IFN-γ secretion in human LAK cells in the presence of human cancer cells. Human LAK cells were incubated with anti-CEACAM-1 antibody (CM-24) at different concentrations at 37 ° C. for 30 minutes. Human lung cancer cells H358 or H460 were then added for a 24-hour incubation. IFN-γ secretion was measured by Elisa. FIG. 11 shows that human LAK cells produce high levels of IFN-γ by themselves, but the addition of anti-CEACAM1 antibody significantly increases IFN-γ levels in a dose-dependent manner.

Example 5. CEACAM1 expression correlates with the presence of B-Raf mutations in cancer cells.
A. By evaluating biopsy samples from 24 melanoma cancer patients for CEACAM1 expression levels and BRAF genotype, there is a statistically significant correlation between B-Raf V600E mutations and CEACAM1 expression. It was revealed. More specifically, only 50% (3/6) of melanoma cells with wild-type B-Raf, ie valine at position 600, express detectable levels of CEACAM1 (36 or less Ct). , That is, 100% (18/18) of melanoma cells having the mutant B-Raf, that is, glutamate at position 600, expressed a detectable level of CEACAM1 (36 or less Ct).

B. CEACAM1 extracellular staining of cancer cells treated with B-Raf or MEK inhibitor. 1.0 × 10 B-Raf W. of ⁶ cells. T. Cell samples (076 mel) and two B-Raf V600E cell samples (526 mel, 624 mel) were incubated with different concentrations of vemurafenib or selmethinib (0.1 μM or 1 μM) for 2 to 48 hours. At each time point, CEACAM1 expression on these cells was determined by FACS. An equal amount of DMSO (vehicle) was used as a control. The results show that 0.1 μM and 1 μM vemurafenib were W. T. Although it did not affect CEACAM1 expression levels on cells with B-Raf (076 mel), 0.1 μM and 1 μM vemurafenib had CEACAM1 expression levels on cells with mutant B-Raf (526 mel, 624 mel). It was shown that it had a dose-dependent effect. The results further showed that selmethinib had a similar effect to vemurafenib on cells carrying the mutant B-Raf (526 mel, 624 mel), whereas 1 μM selmethinib was W. T. Significantly reduced CEACAM1 expression levels on cells with B-Raf but mutated N-Ras (sk-mel-2), and 1 μM vemurafenib did not affect CEACAM1 expression levels. Shown. These results further support the regulation of CEACAM1 via the constitutive activation MAPK pathway, which in this case is promoted by the mutant N-Ras but not by the mutant B-Raf.

C. Inhibitor-resistant cancer cells show increased CEACAM1 expression and restoration of MAPK pathway activity. Two vemurafenib-sensitive B-Raf V600E cell samples (526 mel, 624 mel) and vemurafenib resistant cell lines derived from them were incubated with 1 μM vemurafenib for 2 days. Cells were then analyzed for CEACAM1 protein expression by FACS as described above. Vemurafenib-resistant cell lines were generated by gradually increasing the concentration of inhibitor in culture to 0.32 μM. Vemurafenib-resistant cell lines have been shown to express higher levels of CEACAM1 than vemurafenib-sensitive cell lines. MAPK activity was measured by immunoblotting for phosphorylated ERK1 / 2 (pERK, Thr202 / Tyr204), total ERK1 / 2 and actin 24 hours after exposure to 160 nM vemurafenib, vemurafenib sensitive and vemurafenib resistant B-Raf V600E ( 624 mel) Measured in a cell sample. In vemurafenib-sensitive B-Raf V600E cells, vemurafenib almost completely abolished phosphorylation of ERK1 / 2, where MAPK activity was substantially uninterrupted by vemurafenib in vemurafenib-resistant B-Raf V600E cells.

D. Inhibitor-resistant cancer cells upregulate CEACAM1 expression. B-Raf V600E 526mel melanoma cells were cultured in the presence of 1 μM vemurafenib. Cultures were performed in RPMI 1640 supplemented with 1 mM Na pyruvate, 1 mM Pen-Strep, 1 mM L-glutamine, 1 mM non-essential amino acids, and 10% heat-inactivated fetal bovine serum. Initial concentration of vemurafenib was 0.01 of the determined _IC 50 _(0.64nM). Each week, this concentration was doubled to a 5-fold IC ₅₀ (320 nM) to generate vemurafenib-resistant melanoma cells. Cells were then tested for CEACAM1 expression using MRG1 (mouse antibody against human CEACAM1) by flow cytometry as described above. Total RNA was extracted with trizol according to a common protocol and cDNA was made using reverse transcriptase. Vemurafenib down-regulates CEACAM1 expression in B-Raf V600E melanoma cells, which upregulate CEACAM1 expression levels in acquiring resistance to vemurafenib. It is important to note that CEACAM1 levels in B-Raf V600E melanoma cells after developing resistance to vemurafenib are higher than CEACAM1 levels in untreated (vemurafenib naive) B-Raf V600E melanoma cells. ..

E. Inhibitor-resistant cancer cells upregulate both types of CEACAM1 expression. The above vemurafenib sensitive and bemurafenib resistant B-Raf V600E 526mel melanoma cells were tested by qPCR for the type of CEACAM1 overexpressed in acquiring resistance to vemurafenib. The data shown in FIGS. 12A and 12B show that the expression of both types of CEACAM1 CEACAM1-long (12A) and CEACAM1-short (12B) was approximately 3-fold upregulated in vemurafenib-resistant cells compared to vemurafenib-sensitive cells. Indicates to do.

F. B-Raf / MEK inhibitors increase T cell-induced cytotoxicity. Two vemurafenib-sensitive B-Raf V600E cell samples (526 mel, 624 mel) were tested for viability with or without 1 μM vemurafenib in the presence of cytotoxic T cells. Melanoma cells were pre-incubated with 1 μM vemurafenib and then co-cultured overnight with HLA-A2-matched antigen-matched T cells at an effector-target ratio of 5: 1. Cell killing was determined by LDH release. Vemurafenib has been shown to significantly sensitize melanoma cells to cytotoxic T cells. Antibodies to B-Raf / MEK inhibitors and CEACAM1 increase T cell-induced cytotoxicity to cancer cells in vitro.

Example 6. In vivo anti-cancer effect of CM-24 at different doses IV transplantation of 5 × 10 ⁶ melanoma cells (cell line MEL526) into SCID-NOD mice and treatment for 44 days, depending on the treatment group shown in FIG. did. Antibodies were administered twice weekly by IV infusion and 10 x 10 ⁶ TILs were administered IV every 10 days. Mice were sacrificed on day 49, lungs were removed, imaged (FIG. 13A), weighed (FIG. 13B), and lesions were counted (FIG. 13C). FIG. 13A-Digital photograph of mouse lung immediately after removal. FIG. 13B-Tumor weight was calculated by subtracting the lung weight of untreated mice from the average lung weight of different treatment groups. Results represent mean ± standard error of tumor weight in 7-8 mice per treatment group. FIG. 13C-Number of lung lesions in individual mice of various groups; black lines represent the median of the group; lungs with innumerable lesions were scored as 100. Statistical significance between groups was calculated using paired T-tests. * P <0.05, ** P <0.025.

Although treatment with CM-24 in the presence of TIL resulted in robust tumor growth inhibition, as can be seen by lung morphology (FIG. 13A), tumor weight (FIG. 13B) and number of lung lesions (FIG. 13C). Mice treated with IgG controls showed extensive tumor volume and numerous lung melanoma lesions. Only moderate tumor growth inhibition (TGI 47%) was observed in the group receiving human-reactive T cells for melanoma with control antibody (TIL + IgG), but with the addition of CM-24, all tested. Substantial and dose-dependent anti-cancer activity at doses (84%, 87%, 90% and 93% TGI at doses of 1, 3, 6 and 10 mg / kg, respectively) was shown at 6 and 10 mg / kg. There was statistical significance at a dose of kg (Fig. 13B). Digital photographic recordings of the lungs at the end of the assay (FIG. 13A) showed near normal lung morphology in mice treated with CM-24 in the presence of TIL, thus CM-24 in the presence of TIL. Was shown to eliminate malignant cells almost completely.

As expected, some antitumor effect was also observed in the group treated with TIL and IgG controls. However, significant differences were observed when comparing their effects to CM-24 treated mice in terms of the number of TGI, lung lesions and lung morphology.

Assessment of the number of lung lesions revealed a very small number of lung lesions in all mice treated with TIL and various doses of CM-24, and none of these lungs showed more than 10 lesions. .. On the other hand, in the IgG or TIL + IgG groups, some animals showed a high number of lesions (> 100), with median values in these groups significantly higher than CM-24 treated mice (FIG. 13C) (CM-). 100 and 45 in the IgG and TIL + IgG groups; P <0.025) compared to 5, 6, 3 and 2 in the 24 treatment groups.

These effects were observed at concentrations as low as 1 mg / ml, but were statistically significant in terms of lung weight at CM-24 concentrations of 6 and 10 mg / kg.

Special Note: One mouse in the IgG group showed severe morbidity, including limb paralysis and was slaughtered on day 33 (extensive tumor mass was detected in the lung); one mouse in the TIL + IgG group. Died at the end of the assay on day 49 (several lesions were detected); one mouse in the TIL + CM-24 group showed severe morbidity and weight loss. , 39 days slaughtered (no evidence of lesions detected); 3 mice in the PBS group showed severe morbidity during the experiment, slaughtered on day 42, and 2 other mice Was slaughtered on day 48 (widespread tumor mass, including numerous lung lesions, was detected in all mice).

At the end date, a flow cytometry-based assay in combination with the QuantiBrite kit was used to determine CEACAM1 receptor occupancy by CM-24 in human TIL isolated from the lungs of treated mice. Receptor occupancy (RO) values in mice treated with various CM-24 doses can be attributed to a concentration of 1 mg / kg (CM-24 serum level in blood) of 50% RO, greater than 90%. RO was shown at doses of 3, 6 and 10 mg / kg (CM-24 serum levels 0.3, 48.5 and 111 μg / ml, respectively) (FIG. 14). Testing for CEACAM1 RO levels was performed using a flow cytometry-based assay using PE-bound CM-24 antibody and QuantiBrite kit (BD). Results represent mean ± standard error of RO values in TIL isolated from the lungs of 8-9 mice per treatment group. Data were analyzed using Kaluza software.

From the above data, it is clear that substantial tumor growth inhibition was observed in CM-24 treated mice in the presence of TIL, which resulted in near complete elimination of malignant cells. All CM-24 doses showed a valid anti-cancer response, while increasing doses showed higher values of tumor growth inhibition (84, 87, 90 corresponding to doses of 1, 3, 6 and 10 mg / kg, respectively). And 93 TGI), which was also statistically significant. In vitro RO results showed RO> 90% at doses of 3, 6 and 10 mg / kg (CM-24 serum levels 0.3, 48.5 and 111 μg / ml respectively), with 50% RO. It was detected at a dose of 1 mg / kg. When evaluating RO data from mice treated with CM-24 at a dose of 1 mg / kg, the serum concentration of CM-24 was very low, but the RO data showed that CM-24 still bound to TIL in the lungs. It suggests that CM-24 can mediate long-lasting effects in vivo in the tumor microenvironment.

These results support the mode of action of CM-24 as an enhancer of the cytotoxic activity of effector cells against malignant cells, indicating that CM-24 is a potent anticancer agent.

Example 7. Summary of Preclinical Data CM-24 is a humanized IgG4 anti-human CEACAM1 monoclonal antibody that binds to the N-terminal domain of CEACAM1 and blocks the intercellular CEACAM1 interaction between activated lymphocytes and tumor cells; Blocking CEACAM1 interaction by CM-24 is postulated to enhance lymphocyte killing activity and is a promising tool for the pursuit of immunotherapy for cancer.

CM-24 exhibits high affinity and selective binding to human CEACAM1 expressed by activated lymphocytes or tumor cells. Data from an in vitro immunomodulatory model show that CM-24 is a potent blocker of intercellular CEACAM1-CEACAM1 interactions and varies from CEACAM1-positive NK cells and lymphokine-activated killer (LAK) cells as well as tumor-infiltrating lymphocytes (TIL). It has been demonstrated that it can enhance the cytotoxic killing of human CEACAM1-positive tumor cells. As demonstrated in various models, the CM-24-induced enhancement of killing activity can be mediated by granzyme B and IFNγ secretion.

CM-24 enhances the cytotoxic activity of effector cells in the presence of CEACAM1-positive tumor cells and in the context of a T cell response restricted by a particular human leukocyte antigen (HLA). CM-24 does not enhance the cytotoxic activity of effector lymphocytes against CEACAM1-positive non-target cells (normal human cells). In addition, this data shows that CM-24 has no ligand-like agonist effect, Fc-related effector function or direct effect. Binding of CM-24 to CEACAM1 did not induce agonist activity, and no effect of CM-24 on effector cells could be observed in the absence of target cells, as shown in the in vitro function assay. Since IgG4 cannot bind to complement components C1q and ADCC mediator-Fcγ-RIIIa and does not directly affect the proliferation or survival rate of CEACAM1-positive primary cells (lymphocytes, epithelial cells and endothelial cells), CM-24 also It is expected not to induce antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cellular cytotoxicity (CDC).

Various in vivo tumor xenograft models show that CM-24 has distinct anti-cancer activity with enhanced immunological activity of T cells within the tumor.

CEACAM1 inhibition reduces CEACAM1-mediated inhibition of CEACAM1-positive tumor-infiltrating lymphocytes that encounter CEACAM1-positive cancer cells within the tumor microenvironment. The immunological effect is expected to be an enhancement of the local immune response to the tumor cells, leading to the removal of the tumor cells and subsequent clinical regression.

Preclinical evaluation of the safety of CM-24 included evaluation of the effect of CM-24 on T cell proliferation and pro-inflammatory cytokine secretion. Human peripheral blood mononuclear cells (PBMC) and whole blood from 10 healthy donors were evaluated and the antibody was provided in both soluble and immobilized form. There was no overt CM-24-induced proliferation and significant pro-inflammatory cytokine secretion in soluble or dry-coated immobilized stimulus forms.

A 6-week repeated dose study was performed on rhesus monkeys to evaluate the toxicity and toxic kinetics of CM-24. CM-24, a total of 4 doses (intended in tumor patients), representing 2.5 times (25 mg / kg) and 10 times (100 mg / kg) the maximum dose (10 mg / kg) planned in humans. (Mimicking the treatment cycle) was administered biweekly via intravenous (IV) injection (the intended clinical route of administration). No obvious treatment-related side effects, macroscopic or microscopic pathological findings, or death were observed. Ophthalmoscopic examination and electrocardiogram showed no treatment-related findings. In addition, whole blood and urinalysis were found to be within the normal range of rhesus monkeys. Taken together, a significant GLP repeated dose toxicity study in rhesus monkeys showed no treatment-related toxicity at all dose levels and a no-effect dose (NOEL) was determined to be 100 mg / kg. In the case of administration by IV infusion, toxicological assessment showed a dose-proportional increase in CM-24 exposure (Cmax, AUC). A slight increase in Cmax and AUC on day 42 compared to day 0 provides some potential for CM-24 accumulation when mAbs are administered by this ROA and dosing regimen at these dose levels. It may suggest. Since only one animal in Group 2 (25 mg / kg) showed a positive anti-CM-24 antibody response, it is most likely that the ADA response has no effect on either pharmacokinetics or toxicity studies. It was suggested that it was high. This result does not represent the result of CM-24 and a strong immunogenic response to repeated doses.

The safe clinical starting dose for MABEL-based CM-24, determined from in vivo experiments in the results of a mouse tumor xenograft model, ranges from 0.2 to 1 mg / kg, including a 10-fold factor of safety. Important GLP repeated dose toxicity studies in rhesus monkeys showed no treatment-related toxicity at all dose levels, with a no-effect dose (NOEL) of 100 mg / kg (100 mg / kg weight-to-weight basis human equivalent (HED)). ), Which clearly supports the range determined by the MABEL assessment. Assay results for PBMC proliferation and pro-inflammatory cytokine production in vitro / exvivo showed no substantial CM-24-related induction of T cell proliferation and pro-inflammatory cytokine production.

Example 8. Phase 1, open-label, multicenter, multi-dose escalation study of CM-24 in subjects with selected advanced or recurrent malignancies 6 tumor types-melanoma, non-small cell lung adenocarcinoma, gastric cancer , Colon-rectal cancer, bladder cancer and ovarian cancer-these have high medical needs for new treatments and precedent for clinical responses to other immunotherapies, suggesting that the CEACAM1 pathway is important for tumor progression. It was selected for this study because it is representative of tumors with supportive correlated pathological data.

The trial includes two phases: a dose escalation portion and an expanded cohort portion. The dose escalation portion begins with 4 infusions (cycle 1) for each subject and lasts at least 12 weeks. Subjects (complete response-CR, partial response-PR, stable disease-SD) and diagnostic imaging that are otherwise clinically stable but lack dose limiting toxicity (DLT) and show evidence of clinical benefit Subjects with progressive disease-PRD in the assessment are treated for up to a total of 5 additional cycles over a maximum of 38 additional treatment weeks and at least 25 weeks of follow-up (75 weeks / approx. 15 months total). (Continuous treatment period). The enlarged area covers up to 46 weeks of treatment and at least 25 weeks of follow-up in subjects with cutaneous melanoma (total 71 weeks / about 14 months.

Main purpose:
In subjects with advanced or recurrent malignancies, including melanoma, non-small cell lung adenocarcinoma, and gastric cancer, colorectal cancer, bladder cancer and ovarian cancer.
1. 1. Evaluation of safety and tolerability of multiple doses of CM-24 (intravenous administration), and 2. Determination of recommended Phase 2 dose of CM-24

Included in secondary objectives:
1. 1. Characterization of multiple dose pharmacokinetic profiles of CM-24.
2. 2. Immunogenicity characterization of CM-24.
3. 3. Assessment of preliminary efficacy based on objective tumor response and duration of response in subjects treated with CM-24.

Included in exploratory purposes:
1. 1. Search for potential predictive biomarkers associated with CM-24 clinical activity based on CEACAM1 expression levels in pretreatment tumor specimens.
2. 2. Investigation of the immunomodulatory activity of CM-24 in selected immune cell populations and soluble factors in tumors and peripheral blood.
3. 3. Evaluation of overall survival of subjects treated with CM-24.

Dose escalation portion: Study drug dosing starts at 0.01 mg / kg and continues to 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, and 10 mg / kg. Scheduled to be administered in a shift format. Subjects are assigned dose levels in order of study participation. For the first two dose levels (0.01 mg / kg and 0.03 mg / kg), one patient was enrolled in an accelerated design in each cohort, in which a single grade 2 drug-related toxicity was Extend to 3 + 3 design at that dose and all subsequent doses. For subjects in the two lower cohorts (0.01 mg / kg and 0.03 mg / kg), the first patient's single low-dose cohort (0.01 mg / kg) to the next cohort (0.03 mg / kg) ), And dose escalation from the second single-dose cohort (0.03 mg / kg) to the next cohort (0.1 mg / kg) is a 6-week DLT if no grade 2 or higher toxicity has occurred. Starts after the window. For dose levels of 0.1 mg / kg and above, at least 3 patients per cohort are enrolled in the standard 3 + 3 design, in which case the cohort is 6 patients, unless DLT occurs. Is expanded to. Graduation to the next cohort begins with the first study drug administration of the first subject in each cohort and begins only after an 8-week DLT window.

Dosing period includes 3 periods: screening, dosing and follow-up: 1) 4 repeated doses containing the first cycle followed by a 6 week observation period), 2) continuous treatment period (cycle 2) ~ 6) and 3) Follow-up period including assessment of overall survival. Each treatment cycle consists of 4 doses of study drug administration every 2 weeks. At least three subjects per cohort are enrolled in a standard 3 + 3 design. The minimum planned number of subjects enrolled in this section is 17, but can be increased in the event of dose-limiting toxicity (DLT). If DLT occurs at a constant dose level, this dose level is extended to 6 subjects, thus the maximum number of subjects in the dose escalation portion is 42.

Enrolled subjects receive treatment four times every two weeks (cycle 1), followed by a six-week observation window; at which time the appropriate subject enters the continuous treatment period and an additional cycle (cycle 1). Receive 2-6). During this continuous treatment period, subjects will receive physical examination (weight, vital signs and oxygen saturation), pharmacokinetics and pharmacodynamics, cytokine collection, as well as clinical and laboratory evaluations including safety laboratory tests and immunosafety assays. And the ECG is recorded. All subjects undergo a 1-week and 5-week response assessment after cycle 1 based on diagnostic imaging and clinical assessments. If qualified to continue the test, an additional response assessment will be performed shortly before the start of the next cycle. After the last continuous treatment cycle, subjects are observed for safety, efficacy and survival.

Enlarged part: Up to 20 metastatic cutaneous melanoma subjects are enrolled and treated with the recommended Phase 2 dose (RP2D). This includes Phase 3: screening, medication and follow-up. The dosing period consists of 6 cycles of 4 treatments, each given every 2 weeks. The follow-up period includes assessing overall survival.

Enrolled subjects receive the recommended Phase 2 dose every two weeks in each cycle. Four treatments are given and the subject continues treatment for up to 6 cycles. As detailed above, during the dosing period, the subject undergoes clinical and laboratory evaluations.

Subjects were all previous chemotherapeutic agents and immunomodulators (eg, but not limited to: anti-CTLA4 antibody, anti-PD-L1 antibody, anti-PD-1 antibody, for at least 4 weeks prior to administration of the first study drug. Must have a "washout" period from laboratory agents except IL-2), must have a "washout" period of at least 6 weeks prior to administration of the first study drug, and all adverse events , Returned to baseline or stable below grade 1.

Melanoma subjects with B-Raf V600E or V600K mutation-positive melanoma had to be advanced or intolerant to B-Raf inhibitor therapy and / or MEK inhibitor therapy.

Dose escalation Part Cycle 1 consists of a 6-week repetitive dosing period (4 doses, each with a 2-week interval) and a 6-week observation period.

A minimum of one week must elapse between the initial treatment of any subject in the dose cohort and the initial treatment for subsequent subjects in that dose cohort.

Subjects will be carefully observed during the 12-week initial study period. All subjects underwent response assessment 1 and 5 weeks after the end of cycle 1 for evidence and confirmation of clinical benefit, and by 12 weeks stable disease (SD), partial response (PR) or complete response (CR) Is defined as. Subjects who had evidence of progressive disease (PRD) on either 7th or 11th week of imaging after regaining consent with informed consent (ICF) were included in the clinical exacerbation section. If the investigators believed that it was clinically guaranteed under the discontinuation rules, they could continue to participate in the study, continue CM-24 treatment, and were evaluated at 15 weeks. If the 15-week follow-up imaging shows PRD, the subject will not continue treatment due to confirmed disease progression.

All subjects with DLT, including delayed DLT, will be discontinued from further dosing with CM-24, but the study will not be discontinued.

Subjects who have been discontinued before the first three study drug administrations are completed during the initial study period (cycle 1) will be replaced. Subjects who have been discontinued for any reason other than withdrawal of consent will be observed on more than four follow-up visits for a period of 6 months.

Cycles 2-6 are called continuous treatment periods. Subjects who have evidence of clinical benefit defined as SD, PR or CR according to modified RECIST 1.1 criteria after the initial study period and no evidence of dose limiting toxicity (DLT) by 12 weeks During the 5 additional treatment cycles, CM-24 treatment at the same dose level received during the initial study period can be continued. Subjects with confirmed but not aggravated PRD and otherwise stable or improved clinical status should continue treatment with study drug until further progression or worsening of clinical symptoms. is there.

Each complete treatment cycle during the continuous treatment period comprises four doses of study drug administered at two week intervals on days 1, 15, 29, and 43. Response assessments are performed between days 52 and 56 of each treatment cycle. Response assessment must be completed before the first dose of the next cycle is administered.

During the duration of continuous treatment, subjects with confirmed but not aggravated PRD, and otherwise stable or ameliorated clinical conditions, have further progression or worsening of clinical symptoms. Until then, treatment with study drug should be continued.

After the last dose of CM-24 during the continuous treatment period, each subject will be observed on more than 4 follow-up visits for a period of 6 months.

All subjects with DLT, including delayed DLT, discontinued further dosing of CM-24, but the study was not discontinued.

All subjects are observed indefinitely for survival.

Cohort Expansion For the first two dose levels (0.01 mg / kg and 0.03 mg / kg), one patient was enrolled in the accelerated design in each cohort, in which a single grade 2 drug-related toxicity. Is expanded to a 3 + 3 design at that dose and all subsequent doses. For subjects in the two lower cohorts (0.01 mg / kg and 0.03 mg / kg), the patient's first single low-dose cohort (0.01 mg / kg) in the absence of grade 2 or higher toxicity. Dose escalation from to the next cohort (0.03 mg / kg) and from the second single dose cohort (0.03 mg / kg) to the next cohort (0.1 mg / kg) begins after a 6-week DLT window. .. For dose levels of 0.1 mg / kg and above, at least 3 patients per cohort are enrolled in the standard 3 + 3 design, unless DLT occurs, in which case the cohort is expanded to 6 patients. To. Graduation to the next cohort begins with the first study drug administration of the first subject in each cohort and begins only after an 8-week DLT window.

If additional subjects in the cohort of 6 subjects do not have DLT, dose escalation may proceed after review of safety data for all subjects by the Safety Commission.

If more than one subject develops DLT in a 3 or 6 subject dose cohort, dose escalation is stopped and:
If the previous dose level cohort had not already been expanded to 6 subjects, then expanded to 6 subjects,
If the previous dose level cohort had already been expanded to 6 subjects, the Safety Commission will review all safety data to date after reviewing.
The (previous) dose level can be considered as the recommended Phase 2 dose (RP2D), or evaluation of a new cohort can be recommended at intermediate doses.

The recommended Phase 2 dose (RP2D) is defined as the highest dose level at which less than 1 in 6 subjects will experience DLT.

Dose escalation If neither DLT encounters a 6-week DLT window for the two lower cohorts and an 8-week DLT window for the remaining cohorts, the next cohort is initiated and the same pattern Repeated. There is a one week waiting period between the enrollment of subjects within the dose level of each cohort. Prior to dose escalation, available safety data for all subjects treated in previous studies, including the current cohort, will be evaluated by the Safety Commission.

Dosing of the next dose cohort (cycle 1) is initiated only after repeated dosing (cycle 1) for all subjects in the previous dose cohort is completed. The timing of the Safety Commission review is based on data suggesting that irAE occurs within 5-10 weeks after dosing in patients treated with other immunomodulators. When DLT occurs and the therapeutic dose cohort needs to be expanded to 6 subjects, the DLT window is expanded to include complete repetitive dosing (cycle 1) for all 6 subjects.

After dose escalation, if more than one delayed DLT occurs at a lower dose and AE can be a delayed DLT that may be associated with CM-24, spontaneous increase is temporarily interrupted and the Safety Commission You will be notified within 24 hours. The Safety Commission will promptly evaluate the event and relevant information, including PK data, and make any necessary adjustments in a dose and / or dose escalation scheme. The same procedure is performed if two or more delayed DLTs occur within an expanded cohort of six subjects previously tested.

Delayed DLTs are reviewed by the Safety Commission in accordance with the guidelines and decisions are made on a case-by-case basis. For such behavior, apply standard DLT escalation rules when the dose-related event being tested is not serious enough to stop the dose escalation and the current medication is not considered to be a risk to the subject. It may include doing, returning to a lower or intermediate dose, or taking no action.

Enlarged portion Up to 20 subjects with advanced cutaneous melanoma can be enrolled in the enlarged portion of this study. If the initial efficacy signal warrants this, the protocol may be amended with other expansion arms of up to 20 subjects deployed in the indications previously investigated during the dose escalation portion of the trial. .. Enrolled subjects are treated with RP2D for up to 6 cycles and are treated on days 1, 15, 29 and 43. Reaction evaluation is performed between days 52 and 56. This response assessment must be completed prior to administration of the first dose in the next cycle.

If the DLT rate is 33% or higher, the registration may be retained in the expanded portion. Subjects who are tolerated by the study drug are not automatically excluded from continuous administration until reviewed by the Safety Commission, and unless otherwise instructed as a result of the safety review, continue medication as long as it is tolerated. Is possible. After the safety analysis, enrollment will be resumed to determine whether to continue dosing at the current dose or at a lower dose to continue enrollment for further subjects. In the case of DLT, registration will be done and / or resumed according to the review of the Safety Commission.

Treatment Decision Guidelines Tumor responses are assessed using response criteria in solid tumors (RECIST 1.1). Completion of the cycle tumor response assessment for all subjects occurs within 52-56 days of each treatment cycle (assess results must be reviewed and documented prior to the first dosing of the next cycle). Subject in additional cycles of CM-24 after each (continuation or extension) treatment cycle, unless the subject develops a Grade 3 or higher (CTCAE) adverse event or other adverse event associated with CM-24 that interferes with further treatment. The decision to treat is based on tumor assessment. Subjects are treated until a complete response (CR) or confirmed and then progressive progressive disease (PRD) is confirmed, as described below. Subjects with confirmed but not deteriorating PRD, and those who do not have a stable or improved clinical condition (ie, no reduction in ECOG general condition) are described in more detail below. As per, treatment with study drug should be continued until further progression or exacerbation of clinical symptoms.

Subjects with the best overall effect (BOR) of CR, PR or SD will continue to receive CM-24 treatment until one of the following occurs first:
Achievement of confirmed CR;
Exacerbation of clinical symptoms suggesting that further benefits from treatment are unlikely;
Meet the criteria for discontinuation of study treatment (DLT) or other intolerance to treatment; or receive the maximum number of cycles.

Follow-up period Subjects will be observed for a period of at least 6 months starting with the last treatment infusion. The first follow-up visit takes place within 7 days of the last imaging scan. The second to fourth follow-up visits are made at intervals of 56 days. In addition, survival is assessed indefinitely approximately every 3 months after completion or discontinuation of treatment and by either telephone call or face-to-face contact during the follow-up of the study. The date of death is reported for all deceased subjects. Overall survival is assessed until the sponsor completes or ends the study. Other data other than survival (eg, subsequent treatment, general condition, etc.) are not collected at the time of these calls or visits.

If the subject discontinues study drug treatment, the date and reason for study drug discontinuation should be documented in the original document and the subject should enter the follow-up period. When a subject discontinues a study (during treatment or follow-up), all assessments related to the study visit will be performed to ensure that the date and reason for discontinuation are documented in the original document. All efforts should be made.

After completion of the treatment and follow-up period, all surviving subjects will be observed indefinitely for survival status every 3 months.

Physical Properties of Test Agent CM-24 is supplied in a disposable 10 mL vial. Each vial contains a concentrate of CM-24 (10 mg / mL) equivalent to 100 mg.

CM-24 is administered as an intravenous infusion at a protocol-specified dose using a 0.2 micron inline filter.

Instructions for preparing different doses:
For subjects receiving doses of 0.1 mg / kg, 0.3 mg / kg and 1.0 mg / kg, a 50 mL 0.9% sodium chloride IV bag is used for preparation. The infusion should proceed at a rate of 1.0 mL / min. Rounding during dose preparation should be performed only when absolutely necessary and only in a manner that allows the minimum concentration of 0.25 mg / mL to be maintained.

For subjects receiving a dose of 3 mg / kg, a 100 mL 0.9% sodium chloride IV bag is used for preparation. The infusion should proceed at a rate of 2.0 mL / min. Rounding during dose preparation should be done only when absolutely necessary.

For subjects receiving a dose of 10 mg / kg, a 250 mL 0.9% sodium chloride IV bag is used for preparation. The infusion should proceed at a rate of 3.0 mL / min. Rounding during dose preparation should be done only when absolutely necessary.

Pharmacodynamic (PD) markers Blood samples are available at the following time points: before dosing the first study drug, 48 hours after the first and fourth dosing of the test drug in cycle 1, each complete cycle (dose escalation) Cycles 2-6, and the entire cycle of the extended cohort), taken before dosing the 4th (last) dosing, during the 2nd to 4th follow-up visits, tested for immunoassays and other assessments, including: Includes:
-Lymphocyte subtypes (CD4, CD8 and CD56) combined with activation markers (CD69, CD107a and HLA-DR) by fluorescence activated cell selection (FACS), CEACAM1 expression in regulatory T cell / lymphocyte subtypes -Soluble CEACAM1 and Granzyme B in serum
-CEACAM1 receptor occupancy by CM-24-Bone marrow-derived suppressor cells (MDSC) (CD14 +, low HLADR, CD11b +)
-Immune checkpoint proteins such as PD-1, TIM-3, LAG, Vista

Pharmacokinetics (PK)
Pharmacokinetics are first tested during the dose escalation portion during the first infusion (first dosing in cycle 1) and the fourth infusion (last dosing in cycle 1). Pre-medication levels are also measured before each treatment during the first cycle. Additional dose-enhanced subjects (up to 6 individuals) may be tested for PK characterization with preliminary RP2D if a stronger PK characterization of CM-24 is justified.

The PK profile of CM-24 in plasma is assessed by 15 days post-dose for the first dose and 36 days post-injection for the fourth dose. The following PK parameters: C _max , t _1/2 , T _max , AUC _0-t , and AUC _0-∞ are derived from plasma concentrations relative to the time profile.

Samples are taken at the time of the first and fourth injections, at the following times: before injection (baseline); at the end of injection and 1 hour, 4 hours, 8 hours, 24 hours after injection. For the first and fourth medications only, the third, fifth, eighth, and fifteenth days (or before the next treatment), and for the fourth medication, an additional 22 days after injection. And the 36th day. Pre-medication levels are measured prior to each treatment during the first cycle. For more details on sample collection and sample shipment, please refer to the event schedule and experimental manual.

Fresh Tumor Biopsy Tumor samples are evaluated for CEACAM1 receptor occupancy by CD4, CD8, CD56, FOXp3, CEACAM1, Granzyme B, CM-24, CM-24:

Dose escalation portion: Subjects are samples of any fresh biopsy tissue (or recorded if taken within the last 6 months) at baseline and 1 week after the second treatment in the first cycle. Is required to provide.

Enlarged portion: Two fresh tumor samples are taken at the time of screening and one week after the second dosing in the first cycle. In addition, the subject is prompted to provide an additional biopsy one week after the fourth treatment in the first cycle, but does not need to.

Effective Endpoints The following endpoints are used to assess preliminary effectiveness and are derived from modified RECIST 1.1 criteria:
・ Objective response rate (ORR)
・ Duration (DOR)
・ Tumor reaction status ・ Disease control rate (DCR)
-Durable Response (DR)
Best Overall Effect (BOR) -Complete Response (CR), Partial Response (PR), Stable Disease (SD) and Progressive Disease (PRD)
-Progression-free survival (PFS)
・ Time to reaction (TTR)
・ Overall survival (OS)
Tumor volume change rate (PCTB) as assessed by CT or MRI.

The effectiveness endpoints listed above are evaluated in the following ways:
Dose escalation cohort:
• At week 7, it was administered 1 week after the 4th dosing in the 1st cycle.
• At week 11, it was administered 5 weeks after the 4th dosing in the 1st cycle.
• 18-19 weeks, 26-27 weeks, 34-35 weeks, 42-43 weeks, 48-49 weeks and 50-51 weeks after an additional 5 cycles, plus a second to fourth follow-up visits.
Dose expansion cohort:
7-8 weeks, 14-15 weeks, 22-23 weeks, 30-31 weeks, 38-39 weeks, 46-47 weeks, plus 2nd-4th follow-up visits.

Immune-Related Efficacy Endpoints Additional preliminary efficacy assessments include the application of immune-related response criteria (irRC) based on changes to RECIST 1.1 (also known as irRECIST) and may include the following endpoints:
-Immune- related best overall effect (irBOR) in reaction categories irCR, irPR, irSD, irPD;
-Objective immune-related response rate (irORR) during the entire study period;
-Ir duration of response (DOirR) for those subjects with an ir- reaction;
• An irORR based on the irBOR results during any number of cycles can also be derived.

Pharmacokinetics (PK) Endpoint Pharmacokinetics is tested during the dose escalation portion during the following PK time points: Samples are tested at the first and fourth infusions, at the following time points: Pre-injection (baseline) Collected at the end of injection and 1, 4, 8 and 24 hours after injection. For the 1st and 4th medications, the 3rd, 5th, 8th and 15th days (before the next treatment), and for the 4th medication, 22nd and 36th after the injection. Day. Pre-medication levels are measured prior to each treatment during the first cycle.

Additional dose-enhanced subjects (up to 6 individuals) may be tested for PK characterization with preliminary RP2D if a stronger PK characterization of CM-24 is justified.

Example 7. Formulations Typical formulations of humanized mAbs of the invention include:

Claims (19)

Specific recognition of human CEACAM1
(I) A heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32.
(Ii) A light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.
A humanized monoclonal antibody (mAb) or an antigen-binding fragment thereof. The humanized mAb of claim 1, comprising a heavy chain variable region sequence having the amino acid sequence set forth in SEQ ID NO: 32 and a light chain variable region sequence having the amino acid sequence set forth in SEQ ID NO: 34. The humanized mAb according to claim 1 or 2, comprising a light chain kappa isotype and a heavy chain selected from the group consisting of IgG4 isotypes and IgG1 isotypes. The humanized mAb according to any one of claims 1 to 3, comprising the light chain set forth in SEQ ID NO: 52. The humanized mAb according to any one of claims 1 to 4, comprising the heavy chain set forth in SEQ ID NO: 53. An isolated polynucleotide encoding the humanized mAb or antigen-binding fragment thereof according to any one of claims 1 to 5. A plasmid comprising the isolated polynucleotide according to claim 6. A pharmaceutical composition comprising a therapeutically effective amount of a humanized mAb or an antigen-binding fragment thereof according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition according to claim 8, for use in the treatment of diseases or disorders associated with the expression, activation or function of the CEACAM protein. The pharmaceutical composition according to claim 9, wherein the disease or disorder is a cell-proliferating disease or disorder. The cell proliferative disorders or disorders include melanoma, colorectal cancer, bladder cancer, lung cancer, non-small cell lung cancer (NSCLC), non-small cell lung adenocarcinoma (NSCLA), gastrointestinal cancer, pancreatic cancer, breast cancer, prostate. The pharmaceutical composition according to claim 10, which is a cancer selected from the group consisting of cancer, thyroid cancer, gastric cancer, ovarian cancer, myeloma and uterine cancer. (I) The humanized mAb according to any one of claims 1 to 5, and a basic amino acid of 1 to 10 mg / ml;
(Ii) The humanized mAb according to any one of claims 1 to 5, and 10 to 100 mg / ml sugar;
(Iii) The humanized mAb according to any one of claims 1 to 5, and a surfactant of 0.01 to 1 mg / ml;
(Iv) The humanized mAb according to any one of claims 1 to 5 of 1 to 50 mg / ml, 4 to 6 mg / ml basic amino acids, 70 to 100 mg / ml sugar and 0.1 to 1 mg / ml. ml of non-anionic surfactant; or (v) humanized mAb according to any one of claims 1 to 5 of 10 mg / ml, 4.65 mg / ml L-histidine, 82 mg / ml sucrose, And 0.20 mg / ml polysolvate 20
The pharmaceutical composition according to claim 8. The pharmaceutical composition according to claim 8, further comprising at least one additional immunomodulatory substance. A pharmaceutical composition for use in the treatment of cancer, comprising the humanized mAb or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the treatment comprises administration of an additional immunomodulatory substance. A pharmaceutical composition comprising. A pharmaceutical composition for use in the treatment of cancer, comprising an immunomodulator, said treatment comprising administration of the humanized mAb or antigen-binding fragment thereof according to any one of claims 1-5. , Pharmaceutical composition. The immunomodulator, anti-human programmed cell death protein 1 (PD-1), anti-PD-L1 antibody and anti-PD-L 2 antibody, activated cytotoxic lymphocytes, lymphocyte activator, and RAF / The pharmaceutical composition according to any one of claims 13 to 15, selected from the group consisting of MEK pathway inhibitors. A diagnostic composition comprising at least one humanized mAb or an antigen-binding fragment thereof according to any one of claims 1 to 5. The diagnostic composition according to claim 17, which is used in a method for diagnosing cancer in a subject in need thereof.
The method comprises contacting the biological sample from the subject or the biological sample obtained from the subject with the diagnostic composition.
Complex formation that exceeds a predetermined threshold is an index of cancer in the subject.
Diagnostic composition. A method for determining the expression of CEACAM1 that contacts a biological sample with the humanized mAb or antigen-binding fragment thereof according to any one of claims 1-5 and the level of immune complex formation. Measuring and methods including.

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